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1
Sensitive detection of deoxyribonucleic acid at ultra low concentrations by SERRS. Graham, D. (University of Strathclyde, Glasgow, United Kingdom).
Genetic research programs such as the human genome project have demonstrated the need for selective detection of nucleic acids at ultra low concentrations (1,2). Current detection techniques require amplification of the genetic material by the polymerase chain reaction (PCR) and detection of the amplified product by fluorescence. A new accurate and reliable method for DNA detection has been developed based on adsorption of DNA on colloidal silver and subsequent signal detection using surface enhanced resonance Raman scattering (SERRS). Improved surface and aggregation chemistry, utilizing modified DNA and a novel aggregating agent, has resulted in acceptable reproduciblity for biological applications. The increased sensitivity circumvents the need for an amplification step and the widespread fluorescence quenching inherent in SERRS greatly extends the range of effective DNA labels by enabling the use of both non-fluorescent and fluorescent chromophores.
Further, compared to fluorescence, much greater selectivity is obtained due to sharp vibrational spectra observed thus reducing the need for separation procedures. Overall, the advantages of the method eliminate the need for time consuming amplification steps and provide a new dimension to labeling chemistry and multiplex analysis of DNA.
(1) McKusick, V. A. FASEB 1991, 5, 12-20.
(2) Cui, X.; Li, H.; Goradia, T. M.; Lange, K.; Kazazian, H. H.; Galas, D.; Arnheim, N. Proc. Natl. Acad. Sci. USA 1989, 86, 9389-9393.
2
Purification and amplification of nucleic acids by solid phase extraction employing hydrated zirconium silicate glass beads and thermophilic strand displacement and amplification (tSDA). Woodard, D. L., Beyer Jr., W. F., Dey, P. G., Jurgensen, S. R., Howard, D. R., McNamara, A. M. (Dept. of Molecular Biology, Becton Dickinson Research Ctr, Research Triangle Park, NC).
Thermophilic Strand Displacement Amplification, tSDA, is an efficient, isothermal method of in-vitro amplification of nucleic acid targets. TSDA methods for the sensitive detection of infectious pathogens such as Mycobacterium tuberculosis and Mycobacterium avium have been reported. To simplify work flow and processing procedures across several different biological matrices, we have developed a technique for the hydration of silicacious material resulting in compounds possessing unique DNA binding properties and characteristics. Using these hydrated materials and 32P labeled DNA, we have determined conditions necessary for optimum binding and near quantitative elution of the radio labeled target. Important factors affecting DNA adsorption to these surfaces have been identified. Significantly, these surfaces quantitatively remove >95% of labeled DNA from aqueous solutions, negating the need for chaotropes. Adsorption of DNA to these surfaces was rapid and avid as multiple water washes failed to elute any labeled target. Following adsorption and washing, eluted DNA was compatible with thermophilic strand displacement amplification. As few as 5 genomic copies of M. tb. could be removed from the bulk solution, washed, eluted and amplified. Application of these surfaces to processed clinical samples resulted in detection of as few as 5 genomic copies and decreased background signals.
3
Long range electron transfer in PNA/DNA duplexes. Koch, T.,
Armitage, B., Hansen, H. F., 
rum, H., Batz, H. G.,
Schuster, G. B. (PNA Diagnostics A/S, Copenhagen,
Denmark).
PNA (Peptide Nucleic Acid) is a DNA mimic which shows high affinity and specificity for complementary nucleic acids. The oligomer is synthesized via peptide synthesis and forms duplexes with DNA which are characterized by a larger pitch and displacement of the base pairs leaving a central "tunnel" in the duplex. Due to their different nature, an examination of the basic properties of PNA/DNA hybrids is a prerequisite for exploring the full potential of the applications of PNA in fields such as biochemistry, diagnostics and medicine.
Long range electron transfer in DNA/DNA hybrids has recently aroused
considerable interest, partly due to the potential application of using
electron transfer in DNA as a novel nucleic acid detection system.
However, the study has been hampered by the synthetic difficulty to
incorporate electron donors/acceptors at fixed internal positions,
whereby the electron transfer is unambiguously related to the 
-stack
of the duplex. By using PNA the relative simple chemical composition of
the PNA monomer offers an additional synthetic flexiblity. Here we
report a novel PNA monomer carrying an anthraquinone. The anthraquinone
monomer was incorporated into a PNA oligomer at a central position and
irradiation of a modified PNA/DNA hybrid leads to electron transfer to
the quinone. Long range electron transfer is detected by oxidative
damage of remote GG doublets and the experiments suggest a mechanism
where the electron migration comprises a series of electron transfer
between the bases, i.e. hole hopping. The intensity of GG damage was
found to be related to base pair complementarity, thus a reduction of
the intensity was seen when a single base pair mismatch was introduced
between the quinone and a remote GG doublet.
4
Natural interferon-
in a hydrophilic gel for the treatment of
intravaginal warts in women; A placebo-controlled, double-blind
study. Syed, T. A.1, Ahmadpour, O. A., Ahmad, S.
A.2 (1Department of Dermatology, School
of Medicine, University of California, San Francisco, CA2Medical Faculty, University of the Punjab,
Lahore - Pakistan).
Interferons have shown to be effective for eliminating genital warts.
This viral infection is caused by, small, nonenveloped, icosahedral DNA
viruses (Papillomaviruses), that replicate in the nucleus of squamous
epithelial tissues, and some of their types have shown to be associated
with cervical dysplasia. The study was aimed to evaluate the clinical
efficacy and tolerability of human leukocyte interferon- (2 x
106 IU/g) in a hydrophilic gel (Hydroxyethyl-cellulose,
1%) to cure intravaginal warts. Fifty patients aged 18-50 years (mean:
26.3) with the clinical/biopsy-proven diagnosis of vaginal condylomas,
mean: 5.2 were randomized to 2 parallel groups. Each patient received a
preoded tube (45-g), with a graduated applicator to inject 4-g of it
deep into the vagina 2 times a day for 5 consecutive days per week,
(max. 4 weeks). Patients were examined on a weekly basis. DNA HPV (-)
confirmed complete regression of lesions was considered healed. By the
end of the medication 44% of patients and 44.2% of lesions were
cured. Broke the covered indicated, that interferon- in gel cured
72% of patients and 74.4% lesions, while placebo cured 16% of
patients and 14.5% of warts, (gel vs. placebo; p <0.001). Of the 50
patients 86% complained of no drug-induced side effects. Seven
patients mostly in the interferon- gel experienced non-objective,
mild headache, tenderness, with transient increase in body (>38 °C).
Study was followed-up for 18 months, 3 had a relapse after 8 months. In
conclusion, the findings showed that along with non-objective mild side
effects, leukocyte interferon- (2 x 106 IU/g) in a
gel, is safe, and more effective than placebo to cure intravaginal
warts in women.
5
Imiquimod (5%) in a hydrophilic cream for the treatment of genital herpes in women; A placebo-controlled, double-blind study. Syed, T. A.1, Ahmadpour, O. A., Ahmad, S. A.2 (1Department of Dermatology, School of Medicine, University of California, San Francisco, CA2Medical Faculty, University of the Punjab, Lahore - Pakistan).
Imiquimod (imidazoquinoline) is a low molecular weight heterocyclic amine that stimulates the production of alpha-interferons and other cytokines, thus enhances or upregulates cell-mediated responses. The objective of this study was to evaluate the clinical efficacy and tolerability of Imiquimod in a hydrophilic cream to cure patients afflicted with the first episodes of genital herpes. Sixty female patients, ranging between 18 and 50 years of age with culture-confirmed herpes simplex genitalis, harboring 612 lesions (mean 10.2) were randomized to 2 parallel groups. Patients joined the study within 6 days (mean 4.1) of the manifestation of lesions. Each patient was given a precoded 45-g tube containing active or placebo preparation with instructions on self-administration of trial medication, at home, to their lesions 3 times daily for 5 consecutive days per week. Patients were examined 2 times a week. A reepithelialized lesion with some residual erythema was recorded as healed. The study lasted for 12 weeks with 2 weeks active treatment. Results indicated that patients treated with Imiquimod cream had significantly shorter mean duration of healing than placebo (5.2 vs. 14 days; p<0.01), and had also cured more patients than placebo, 73.3% vs. 16.7%; p<0.001. Of the 60 patients 52 (86.7%) complained of no drug-related side effects. Physicals and lab. tests were clinically within limits, no hepatic, renal toxicity or other hematological assays exceeded the normal limits. Study was followed-up for 18 months, 4 had a relapse after 11 months. In conclusion, the study demonstrated that along with non-objective mild side effects, Imiquimod (5%) in a hydrophilic cream is more effective than placebo to cure first episodes of genital herpes.
6
Detection of new mutations in alpha globin gene using high resolution fragment analysis and DNA sequencing. Reddy, P. L., Bowie, L. J., and Nowak, E. M. (Dept. of Path. and Lab Medicine, Evanston Hospital, Evanston, IL and Center for Biotechnology, Northwestern University, Evanston, IL).
The most prevalent alpha thalassemia mutation in the US is the - 3.7
deletion. This deletion is caused by unequal crossover in the region
around exon 3 of the 1 globin gene of one chromosome and exon 3 of
the 2 globin gene on the other chromosome. Three subtypes (3.7i,
3.7ii and 3.7iii) have been described based on the position of
the crossover. We have described methods to detect -3.7 deletions by
using F37.1 and F37.3 primers (Clin. Chem. 1994; 40: 2260-6) and also
its subtypes by using primers which bind to exon 3 of the
1 globin gene (Clin. Chem. 1996; 42: 1892). We have now
developed primers (C3 and Dev) specific for exon 3 of the
2 gene in order to detect subtypes. We have used high
resolution fragment analysis to identify the -3.7 deletion and also
its subtypes. We deteced new fragments which were slightly different
from the expected size (eg: 366 bp versus 375 bp with F37.1 and F37.3
primers and 395 bp versus 389 bp with C3 and Dev primers).
DNA sequencing of these fragments demonstrated the presence of three
mutations which had not been described or fully characterized before.
Since these mutations were in the non-coding regions and were
clinically silent, the results also demonstrate the effectiveness of
this approach in detecting silent mutations.
7
Genotyping and primer extension preamplification applications in preclinical and clinical pathology testing. Bleavins, M. R., Gipson, T. S., Duddy, S. K., and de la Iglesia, F. A. (Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research, Warner-Lambert Co., Ann Arbor, MI).
The needs of modern drug development present challenges that are dramatically changing the overall functions of the clinical pathology laboratory. Our laboratories have developed PCR and PCR-RFLP based genotyping tests to assess polymorphisms in human P450-1A1, P450-2D6, P450-2C19, glutathione S-transferase, angiotensin-converting enzyme, and ApoE. These peripheral blood-based procedures allow detection of polymorphisms that lead to over expression, lack of expression, or production of mutant proteins with decreased activity. Application of a panel of these genetic tests assists in designing safe and informative clinical trials, offers opportunities for stratification of volunteers or patients, permits prediction and enhanced monitoring of potential drug interactions, and contributes to the determination of appropriate dosing regimens. Similar methodologies are being developed for laboratory animal species to more effectively characterize species-specific toxic responses in preclinical models of safety evaluation. Molecular analysis of altered genomic DNA also is essential for understanding mechanisms by which drugs induce or modify the development of tumors in carcinogenicity studies. The assessment of microsatellite polymorphisms, loss of heterozygosity, and gene mutations allows specific comparisons to normal tissue, or to corresponding tumors seen in humans. Utilization of these new techniques, however, is frequently hampered by limited availability of tissue. To address these limitations, we have combined whole genome amplification by primer extension preamplification with locus-specific PCR. This approach allows analysis of 10 to 25 genetic markers from as little as 1 mm2 of a formalin-fixed paraffin section. These methods have provided our clinical pathology laboratory with powerful approaches for elucidating molecular events in a dynamic environment that integrates animal and human genetic studies.
8
Limitations of Bordatella pertussis detection in respiratory specimens by polymerase chain reaction, culture and direct immunofluorescence. Wei, Q., Robinson, C., Roe, M., and Todd, J. (Department of Pathology, Children's Hospital, Denver, CO).
A sensitive method for detection of Bordatella pertussis in nasopharyngeal aspirates by PCR (Glare, 1990) was compared to culture and direct immunofluorescence (DFA) during a period of high pertussis circulation. In reconstruction experiments with pooled nasal aspirates, the PCR detected approximately 3 logs more viable organisms than culture and approximately 8 genome equivalents per reaction. Subsequently, 356 clinical specimens submitted for pertussis testing were tested by PCR, culture and DFA. We found it was essential to include an internal control for extraction and amplification with every specimen because >10% of aspirates could not be initially amplified and would have been falsely scored as negative without further processing. With this internal control, the PCR was found to be considerably more sensitive than culture (culture sensitivity = 44% vs. PCR) and DFA (DFA sensitivity = 67% vs. PCR). Most specimens detected only by PCR met the clinical case definition of pertussis, so the specificity of PCR was considered to be excellent. PCR failed to detect only one culture positive specimen, but surprisingly, specimens from 4 adults, all of whom had symptoms compatible with pertussis and had been exposed to confirmed cases, were positive only by DFA. These results suggest that despite our enthusiasm for detection of B. pertussis infections by PCR, careful controls must be used to avoid reporting false negative results and positive specimens from all age groups may not be detected by PCR. We recommend the continued use of DFA together with PCR for detection of B. pertussis infections at our institution.
9
Differential display analysis of interferon induced genes in trisomy 21 cells. Siembieda, S. and Lakatua, D. (HealthPartners/St. Paul Ramsey Medical Center, St. Paul, MN).
Interferon is a potent biological activator of many genes. It can
affect both transcription and translation. Interferon binds to an
/ß interferon membrane receptor expressed from chromosome 21.
Interferon is also constitutively expressed during pregnancy. While the
hypersensitivity of trisomy 21 cells to interferon at the protein level
is well documented, gene expression studies have been difficult to
perform. Differential display analysis (DDA) was used to identify
interferon induced differences in gene expression.
Normal and trisomic 21 amniocytes were obtained and grown in
supplemented Chang's media. The cells were weened to a media
containing heat inactivated FBS to eliminated endogenuous interferon.
Poly A+ RNA was isolated from confluent cultures which were either not
induced or induced with high (1000 units/ml) or low concentrations of
-interferon(10 units/ml). Thirty nanograms of mRNA was reverse
transcribed and an aliquot was used for DDA. Four downstream primers
were used in combination with 12 upstream primers. DNA fragments were
electrophoresed on a nondenaturing acrylamide gel and bands were
observed by silver staining.
High and low interferon concentration were used to mimic physiological
conditions. Forty eight primer combinations were used with each mRNA
isolation to identify the differentially expressed genes. No
differences were observed among any of the mRNA preparation using
-interferon as an inducer of gene expression. These findings raise
the question of specificity of gene expression to interferons. Inducing
these amniocytes with interferons ß and Tau might identify genes
responsible for the Down's phenotype.
10
Parallel production of arrays using microporous supports. Stimpson, D. (Garland, TX).
The number of applications for arrays of binding molecules has increased dramatically in the last few years. Current trends in expression screening, mutation detection, development of cDNA libraries and drug discovery require the availability of arrays with uniform and reproducible binding characteristics. Much of the recent work on array production has centered on the application of reagent "dots" or chemical synthesis of "zones" on flat surfaces and those using glass or silicon substrates are often called "chips". As such, each zone on each chip represents a unique synthesis or reagent application step. The present study explores the use of a microporous material as the array substrate. In one embodiment, reagents were immobilized as lines on a sheet of membrane material. The sheet of membrane was then tightly wrapped around a support tube to form a spiral wound structure with the reagent lines parallel to the tube axis. Replicate arrays were generated by cutting the spiral wound membrane along the tube axis much like cutting slices from a "jelly roll". The freshly cut edges of the membrane display the immobilized reagents as binding zones arranged in a spiral array. Each reagent line forms the coincident binding site for many arrays. Binding and detection was carried out using standard methods developed for "dot blot" systems and data for microparticle and enzyme labels are presented. While the method is relatively new and requires further development, this study indicates that arrays can be produced with 50 x 100 micron size zones. The method is most applicable to producing arrays from pre-synthesized materials and should find use in ASO hybridization and organization of cDNA libraries.
11
Clonal analysis of B-cell lymphoproliferative disorders: Comparison of Southern blot and PCR. Tsongalis, G. J., Stevenson, A., Vadlamudi, G., Hodges, K., Rezuke, W. (Department of Pathology, Hartford Hospital, Hartford, CT).
The rapid and more sensitive features of PCR make it a desirable assay for determining clonality in B-cell neoplasms when compared to the more laborious Southern blot (SB) analysis. However, variable rates of detection have been reported for PCR assays when compared to SB which may be due to the subtype of B-cell neoplasm as well as the type and the number of primer sets used. We evaluated a PCR-based assay for detecting clonality in various subtypes of Southern blot positive B-cell lymphomas. PCR amplification using consensus IgH variable region and joining region primer sequences was performed using only two sets of primers in a total of 110 different cases of B-cell lymphoproliferative disorders which were determined to be monoclonal by SB analysis. Immunoglobulin heavy chain gene rearrangements were detected by PCR in 42% (46/110) of cases which included 14/20 marginal zone/MALT, 10/32 follicle center cell, 8/35 large cell, 3/3 T-cell rich B-cell, 5/9 CLL/SLL, 2/3 small non-cleaved cell, 3/5 lymphoblastic, and 1/3 plasma cell dyscrasias. No rearrangements were detected by PCR in normal control DNA and 11 cases of T-cell lymphoma. PCR determination of clonality in B-cell lymphomas appears to be influenced by subtype of neoplasm with the highest sensitivity for MALT lymphomas (70%) and the lowest sensitivity observed for follicle center lymphomas and large cell lymphomas. PCR provides a rapid and specific diagnostic tool for screening lymphoma specimens for clonality before referral to SB analysis for PCR negative cases.
12
Confirmation of BRCA1 and BRCA2 mutations using the universal GeneComb test kit. Linfert, D., and Tsongalis, G. J. (Department of Pathology, Hartford Hospital, Hartford, CT).
Germline mutations in the BRCA1 and BRCA2 genes bestow increased lifetime risk for hereditary breast and/or ovarian cancers. Several common mutations can be detected using a PCR-mediated, site-directed mutagenesis (PSM) assay, which transforms either the wild-type or mutant allele by adding/removing a restriction endonuclease site with a single base substitution to one of the primers. Genotyping is then determined by electrophoresis of the digested amplicon. DNA was isolated from 49 putative, sporadic breast tumor samples using a non-organic extraction protocol. All tumors were screened for 4 BRCA1 mutations (185delAG, 5382insC, R1443X and E1250X) and 1 BRCA2 mutation (6174delT). Allele specific oligonucleotide (ASO) probes were then used in conjunction with the Universal GeneCombTM Test Kit (Bio-Rad Laboratories, Hercules, CA) to confirm the presence of a mutation. Briefly, ASO probes were spotted onto nitrocellulose teeth of the GeneCombTM card and fixed by UV cross-linking. Biotinylated amplicon was then hybridized to the ASO's by capillary action and a colorometric detection was performed. We identified a 185delAG mutation (BRCA1) and a 6174delT mutation (BRCA2) in the same tumor sample using PSM. Double heterozygosity in this tumor sample was then corroborated by the Universal GeneCombTM Test Kit. All other breast tumors did not show evidence of BRCA1 or BRCA2 mutations. In conclusion, PSM provides a rapid method for identifying specific mutations in the BRCA1 and BRCA2 genes, and the Universal GeneCombTM Test Kit provides a expeditious method to confirm mutation analysis.
13
Direct detection of HBV genomic DNA in plasma using transcription-mediated amplification (TMA). Adams, C., Nelson, N. C., and Mimms, L. (Gen-Probe, Inc., San Diego, CA).
The standard assay for the detection of hepatitis B virus (HBV) in donated blood is an immunoassay for HBV surface antigen (HBsAg), a viral envelope protein and the first serologic marker indicative of HBV infection. The most important limitation of immunoassays is its failure to detect the presence of infectious HBV in blood during the six to eight weeks following HBV infection prior to seroconversion. Therefore, diagnostic tests for the direct detection of HBV genomic DNA have the potential to greatly reduce or even eliminate this window of infectivity in the worlds blood supply. Our objective was to develop a single-tube assay that could detect HBV genomic DNA in donor plasma and could be automated for high-throughput blood screening. We have successfully developed such an assay and it consists of three basic steps: [1] purification of HBV genomic DNA from plasma using magnetic particle separation (sample processing), [2] amplification of target nucleic acids by TMA, and [3] quantitative detection of amplicon levels using the Hybridization Protection Assay (HPA). Unlike lengthy PCR-based assays currently available, this assay does not show any signs of sample inhibition and total assay time is less than 4 hours. Assay sensitivity using Eurohep standards is currently approaching 100 genomes/ml of specimen for both Adw and Ayw strains of the virus. We believe that this assay could provide a means for the high-throughput screening of individual samples of donated blood for HBV infection and, because it is a nucleic acid-based assay, will shorten the window of infectivity by weeks compared to currently employed serologic assays, thus improving the safety of the words blood supply.
14
The simultaneous detection and differentiation of Chlamyida trachomatis and Neisseria gonorrhoeae using transcription mediated ampliciation (TMA). Solomon, M., Carlson, J., Castillo, M., Cheung, J., Light, J., Watson, M., and Shaw, J. (Gen-Probe, Inc., San Diego, CA).
Objective: To develop a diagnostic assay that detects and differentiates both C. trachomatis and N. gonorrhoeae rRNA from male or female urine or swab specimen.
Methods: The specific capture of C. trachomatis and N. gonorrhoeae target rRNA onto magnetic particles from male and female urine or swab specimen (target capture) effectively removes inhibitors of nucleic acid amplification. The target rRNA's are then amplified using TMA. The amplification products are hybridized with two different acridinium ester-labeled oligonucleotides that are each specific for the target organisms. Kinetic differences in the in the light-off reactions of the two labels, following a hybridization protection assay, allow for the deconvolution of the signal, and the detection of the two analytes in a single assay.
Results: Male and female urine samples (n=135) were tested in a blind study. Of the 51 known C. trachomatis positive samples 50 were positive in this assay (98%). Of the 22 known N. gonorrhoeae positive samples 22 were positive in this assay (100%). The remaining 62 samples were reported negatives, of which 61 were also negative in this assay (98.4%). Endocervical swabs (n=55) previously tested with Gen-Probe's PACE 2 assay were assayed. Of the 8 C. trachomatis PACE positive samples, 8 were also positive in this assay (100%), and of the 47 PACE negative samples, 46 were negative (97.9%) for C. trachomatis. The TMA assay was not inhibited by 10% blood in urine at the single organism target level. The equivalent of 1 million cells of various Neisseria species did not cause above normal background signal, and the equivalent of a single Neisseria gonorrhoeae cell could still be detected in their presence.
Conclusion: This sensitive and specific assay is being designed in concert with an instrument that totally automates all the steps of sample processing, amplification, and detection.
15
Positively charged enzymatic fragment generation for the detection of rare point mutations. Prudent, J. R., Smith, L., Arco, D., Kaiser, M., Fors, L., Neri, B., and Lyamichev, V. (Third Wave Technologies, Inc., Madison, WI)
Highly sensitive and selective detection of DNA is of fundamental importance in molecular biology and modern medicine. Assays which require commonly used supplies, equipment and standard techniques will allow for more generalized utilization. To this end, we have developed a family of thermostable structure-specific nucleases in conjunction with chemically modified DNA probes, that can be used in a novel linear signal amplification assay to detect and quantify target DNA or RNA point mutations with a discrimination of greater than 1 to 10,000 and a sensitivity in the sub-attomole range. Specifically, the nuclease recognizes a bifurcation at the target site assembled by a distinct upstream invading oligonucleotide along with a chemically modified downstream DNA probe. The enzyme cleaves the DNA probe yielding a net positively charged DNA fragment. This event occurs repeatedly on the same target (cycles), producing multiple fragments per target. As these fragments are the only positively charged nucleic acid species present in the assay mixture, a very brief and low resolution reverse electrophoretic separation (opposite polarity to that employed for standard nucleic acid separations) is sufficient to completely isolate these fragments from all other nucleic acid based molecules present. The enzyme accepts a wide range of 5'-fragment modifications. Therefore, one or more positive charges may be imparted to the cleavage product by means of positively charged modified bases and/or fluorescent dyes such as Cy3, providing a straightforward and highly sensitive means of detection. Employed in conjunction with recent advances in clinically viable gel electrophoretic formats and the use of either real-time or post-separation fluorescence imaging methods, this Charge Reversal Technology (CRT) provides a simple and rapid approach to highly specific and sensitive nucleic acid diagnostics. To our knowledge, this is the first time that a DNA product possessing a net positive charge has been generated enzymatically. The biophysical chemistry and results of this enzyme based reverse charged electrophoresis multiplex assay, detecting and quantifying mutations of the ras, Factor V Leiden, and GPIIIa genes will be shown. The technology should allow for a multiplicity of sensitive gel free readouts to be applied.
16
Semi-arbitrarily primed polymerase chain reaction analysis of DNA associated with the nuclear matrix during GO and S phase of the cell cycle. Phillips, J. M., and Kaufman, D. G. (Dept. Path. Lab Med., UNC, Chapel Hill, NC).
The nuclear matrix is reported to be the primary site of DNA replication, repair and transcription. Though DNA replication is believed to occur in close proximity to the nuclear matrix, it is unclear if the association of the nuclear matrix with attachment sites in the DNA is static or varies temporally with the cell cycle. The objective of this study was to investigate the temporal association of DNA with the nuclear matrix. Nuclei from synchronized human fibroblasts in either GO or S phase of the cell cycle were digested with DNAse I to eliminate DNA not protected by nuclear matrix. A technique recently developed in our laboratory, Semi-Arbitrarily Primed Polymerase Chain Reaction (SAP-PCR), was used to compare DNAse I resistant DNA isolated from GO or S phase. The SAP-PCR technique is similar to differential display but for genomic DNA. Single oligonucleotide primers, or combinations of primers, are used and low stringency amplification conditions are applied in the first two cycles. Subsequent cycles employ the same primer(s) but switch to high stringency conditions for amplification. The patterns of bands that are generated by this technique have been shown to distinguish different species or individuals within a species. In this study, SAP-PCR was utilized to identify sequences of DNA from the same cells but associated with nuclear matrix at different times during the cell cycle. Nuclear matrix-associated DNA isolated GO and S phase produce nearly identical SAP-PCR fingerprints, however, some differentially amplified bands are observed. Although most segments of DNA amplified by SAP-PCR are associated with nuclear matrix during both GO and S phases, there are some fragments of DNA that appear to be preferentially associated with the nuclear matrix during GO or during S phase. Some of these segments have been cloned and sequence analysis is in progress. Supported by CA 42765.
17
Rapid array and screening methods for high throughput analysis of recombinant DNA clones. Lucas, M., Nanda, S. K. W., Sullivan, B. M. (Nalge Nunc International, Naperville, IL).
HTS of libraries for identification and analysis of genes, target drugs, diagnostics and other applications requires extensive indexing and selection of thousands of clones. Management of a large DNA library is simplified by arraying clones in 96 and 384 MicroWell® plates, followed by simultaneous replication in multiple plates or onto membranes.
We screened a S2T cDNA library constructed in
gt11, containing human ß actin cDNA (in E. coli
Y1090), using the NuncTM Replication System. The clonal arrays
were replicated in MicroWell plates and onto membranes with 96 and 384
pin replicators. Optimal growth and replication of the infected
E. coli is achieved within 18 hours. DNA was immobilized on
nylon membranes and the hybridized with a chemiluminiscent labeled
actin probe. Matching clones were identified, isolated and susequently
analyzed. Replication in MicroWell plates and membranes enabled fast
and easy identification of desired clones. ß actin signal intensity
on membranes after a second screening was sharp and distinct without
background smearing. This data clearly demonstrates the efficacy of
simultaneous array using MicroWell plates and hand-held multi-pin
replicators for high throughput recombinant DNA analysis.
18
A homogeneous CPT assay using fluorescence anisotropy. Bekkaoui, F., Chittaranjan, S., Leung, C., and Bryan, R. (ID Biomedical Corporation, Burnaby, BC, Canada).
A homogeneous CPT assay was developed to detect the presence of the mecA gene in methicillin resistant S. aureus (MRSA). CPT or Cycling Probe Technology is an isothermal gene detection method based on the use of target DNA to catalyze the conversion of chimeric hybridization probe into labeled fragments with the enzyme RNaseH. The probe is a DNA oligonucleotide with a scissile link of four ribonucleotides near the center. When the probe hybridizes to the DNA target, a DNA-RNA duplex is created. RNaseH cleaves the RNA portion of the duplex and the resulting fragments, which are now too short to remain stably hybridized, dissociate from the target. Then a new probe hybridizes to the target starting the cycle again. The homogeneous assay uses fluorescence anisotropy (FA) to probe fragments labeled with fluorescein. The CPT-FA assay is performed with crude lysate preparations of Staphylococcal cells in the presence of RNaseH and the chimeric probe. The cleavage of the probe results in a decrease in FA which is plotted against time, and the slope of the curve is proportional to the concentration of target present in the sample. The absolute slope of a mecA positive sample is approximately five fold higher than the absolute slope of a mecA negative sample. Because this format eliminates the necessity for separating the cleaved probe fragments from the uncleaved probe, the assay is rapid. One sample can be processed in less than 20 minutes. Screening of 55 clinical isolates icluding 22 MRSA, 12 methicillin resistant S. epidermidis (MRSE), 10 methicillin sensitive S. aureus (MSSA), three methicillin sensitive S. epidermidis (MSSE), and 8 borderline resistant S. aureus (BORSA) showed 100% sensitivity and 100% specificity compared with standard susceptibility testing. This method could be easily automated and would provide a rapid and accurate method for gene detection.
19
Picogram sensitivity of specific mRNA quantitation by catalyzed reporter deposition assay on oligonucleotide-immobilized microplates. Hamaguchi, Y.1 and Mitsuhashi, M. 1,2 (1Department of Pathology, University of California, Irvine, CA and 2Hitachi Chemical Research Center, Irvine, CA).
Introduction. We have previously demonstrated colorimetric ELISA (enzyme linked immunosorbent assay) measurement of specific mRNA on oligonucleotide-immobilized microplates with a sensitivity of 100 pg to 10 ng (Tominaga et al. Clin. Chem. 42: 1750-1757, 1996). In this study, various attempts were made to improve its sensitivity.
Methods. Rabbit a -globin mRNA was captured on D(-)-P-hydroxyphenylglycine-treated microplates to which oligo(dT) had been covalently immobilized. The captured mRNA was immediately reverse transcribed into cDNA in the presence of biotinylated dUTP. After streptavidin-horseradish peroxidase conjuates (SA-HRP) were bound to the incorporated biotin, biotinyl-tyramide was added and deposited to the surface of the plate. SA-HRP solution was applied again and colorimetric reation was conducted by addition of substrate (ABTS). Its absorbance was measured at 405 nm.
Results and Conclusion. By treating the microplates with various blocking buffer at different stages during assay, and by optimizing various assay conditions, the sensitivity of original colorimetric mRNA ELISA was improved approximately tenfold. Furthermore, based on catalyzed reporter deposition with overnight hybridization of mRNA, colorimetric signals were improved approximately 100 fold, and total and specific mRNA can be measured from 300 fg - 1 pg.
20
Development of quantitative assays using the 5' nuclease technology: The promise and the challenge. Kwok, S.1, Wang, Z.2, Lu, S.1, Tsang, S.1, Kao, S. Y.1, Weiss, J.1, Wang, H.2, and Spadoro, J.2 (1Roche Molecular Systems, Sommerville, NJ and 2Roche Molecular Systems, Alameda, CA).
Quantitative assays that use the 5' nuclease technology offer exciting capabilities as well as developmental challanges. The technology provides for real-time detection of PCR products by monitoring the increase in fluorescence during the amplification process. The elimination of post-PCR manipulation, the broad detection range, and the high throughput potential makes it ideal for routine diagnostic use. Prototype assays that have a sensitivity of about 10 copies per amplification and a dynamic range of 6 logs have been developed for HCV, HIV, HBV, and CMV. Ninety six samples can be analyzed in 2.5 hours, not including sample preparation time.
Although the technology is promising, development of assays for routine diagnostic use poses several challenges. For example, the efficiency of probe cleavage is directly proportional to the number of mismatches. HIV transcripts that have 3-4 mismatches to the probe are either not cleaved or inefficiently cleaved, despite efficient amplification. The incorporation of a quantitation standard that monitors amplificaiton efficiency is complicated by the "competitive" nature of the amplification of the target and quantitation standard. The identification of separate reporter-quencher molecules that can be easily resolved by available instruments is also required.
As efforts continue in improving mismatch tolerance in the 5' nuclease assay, the lower temperature, longer hybridization times, and less stringent buffers used in the AMPLICOR MONITORTM Test are presently better suited in detecting heterogeneous target sequences.
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A novel rapid multiplexed assay for herpres simplex virus DNA using the FlowMetrixTM cytometric microsphere technology. WalkerPeach, C. R.1, Smith, P. L.2, DuBois, D. B.1, Fulton, R. J.2 (1Cenetron Diagnostics, Austin, TX, 2Luminex Corporation, DeSoto, TX).
Sensitive assays for herpes simplex virus DNA are essential for the accurate diagnosis of herpetic encephalitis. We have developed an assay for HSV using FlowMetrixTM, a rapid, accurate state-of-the-art diagnostic system with true multiplexing capabilities. The assay involves amplification of a region of the HSV gB gene (148 bp product) and capture of an internal region within the amplification product. As a control for internal inhibition of the PCR reaction by factors present in patient samples, an internal plasmid control (plCC4) was constructed. plCC4 is a cloning vector which contains the 148 base pair amplification fragment containing an plCC4-specific sequence within the capture region. The two coamplification products have indistinguishable Tm's, hybridization characteristics and are coamplified with the same primer set.
In the FlowMetrixTM system, two sequence-specific oligonucleotide targets (one for HSV gB and one for plCC4) are individually bound to two subsets of microspheres. The microsphere subsets are distinguishable based on unique levels of incorporated orange and red fluorescent dyes. These microsphere subsets are then mixed to form the multiplexed set. Additionally, two bodipy (green fluorescence) labeled probes, complementary to each of the target sequences are also prepared and mixed. Each of the complementary probe/target pairs represent the internal sequence-specific region within the HSV gB gene and plCC4 control. Upon completion of the PCR reaction, the amplification products are hybridized in a multiplex reaction containing both bopidy-labeled probes and target microspheres. Selective hybridization of the PCR products with the target microspheres defines the amplicon type while, a decrease in bodipy fluorescence indicates quantity of the specific PCR product within the hybridization. The results show that the FlowMetrixTM system is capable of extremely rapid and accurate simultaneous detection of specific PCR products.
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Second generation PCR reagents: Their use in optimizing high GC, multiplex, and long PCR's. Grunenwald, H. and Watson, J. (Epicentre Technologies, Inc., Madison, WI).
As PCR becomes routine in clinical laboratories, the current methodologies for optimizing PCR reactions prove to be laborious, especially in developing protocols that meet the rigors necessary in diagnostic testing. Traditionally, individual components of the PCR reaction were optimized separately in order to achieve suitable specificity and sensitivity for a given amplification. In this abstract, we describe a unique buffer reagent system for faster and more reliable PCR optimization. This system contains a series of twelve PCR reaction buffers which include all the components necessary for amplification with the exception of template, primers and DNA polymerase. Using this PCR buffer system, we were able to reduce the optimization time for i) templates with high GC content, e.g., human apolipoprotein E, Bordetella pertussis insertion sequence 481, and the Thermus aquaticus mutS gene, ii) templates with secondary structural elements such as the human cystic fibrosis transmembrane conductance regulator gene and Vaccinia CAPVAC, iii) multiplex PCR amplifications and iv) long PCR, e.g., up to 40 kb ampified from lambda genome. Since magnesium, buffers, salts and co-solvents are all included in the premix, assay-to-assay variability is minimized. The inclusion of various concentrations of reaction components substantially improves the likelihood of amplification for a given set of cycling conditions. This system often eliminates the tedious chore of running multiple cycling parameters in order to optimize a PCR. This PCR buffer reagent system allows molecular diagnostics laboratories to rapidly develop new assays at a substantially reduced cost.
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Mutation detection and DNA fingerprinting at single nucleotide resolution using base excision sequence scanning (BESS). Hawkins, G. A., and Hoffman, L. M. (Epicentre Technologies Corp., Madison, WI).
Base Excision Sequence Scanning (BESS T-Scan) is a new PCR based mutation detection and DNA fingerprinting methodology consisting of a simple enzymatic digestion and denaturing PAGE analysis. BESS involves PCR amplification of target DNA with a partial substitution of dU for dT. Digestion of end-labeled, dU-substituted PCR product with uracil DNA glycosylase (UNG) and an apyrimidinic-apurinic endonuclease releases a nested set of tagged fragments corresponding to the locations of deoxythymidine residues in the original DNA sequence. The labeled fragments of DNA are resolved by polyacrylamide gel electrophoresis producing a DNA ladder virtually identical to a "T" lane sequencing ladder. The appearance, disappearance or change in intensity of a band compared to a control lane pin points the site of sequence variation. This methodology, BESS T-Scan, requires no optimization beyond the PCR stage, no purification of the PCR product and no special gel electrophoresis conditions. The process lends itself well to automation at several stages. For example, the LI-COR IR2 automated DNA sequencer with its associated software has been used to generate BESS data from several genes. The BESS T-Scan method detects and localizes point mutations, frameshifts, insertions, deletions and repeat expansions. Sequences analyzed by BESS T-Scan include human papillomavirus DNA, bacterial 16S rRNA genes, human mitochondrial DNA, HLA-DQA, and BRCA 1, demonstrating the broad utility of BESS T-Scan for detecting mutations and for DNA fingerprinting.
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Analysis of fetal DNA in maternal plasma: A novel approach for noninvasive prenatal diagnosis. Lo, Y. M. D.1, Poon, P. M. K.1, Wainscoat, J. S.3, Lau, T. K.2, Chang, A. M. Z.1, Hjelm, N. M.1 (1Department of Chemical Pathology and 2Department of Obstetrics & Gynecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N. T., Hong Kong, and 3Department of Hematology, John Radcliffe Hospital, Oxford, United Kingdom).
Fetal cells in maternal circulation is a well recognized phenomenon but the existence of free fetal DNA in maternal plasma has not previously been demonstrated. We aim to prove the existence of circulating fetal DNA in maternal plasma by the amplification of Y-specific sequence from plasma obtained from women bearing male fetuses. We developed a sensitive TaqMan assay for a Y-specific sequence (DYS14) which was used to detect fetal Y-chromosomal sequences from the plasma obtained from 26 pregnant women (gestational ages: 12-40 weeks). DNA extracted from 40 ul of maternal plasma was used per assay. Y-signal was obtained from the 18 women bearing a male fetus and no signal was obtained was obtained from the 8 women bearing a female fetus. These results show that fetal DNA exists in maternal plasma and that certain fetal genetic diagnosis, e.g. sex determination, may be made reliably using this approach.
Our next goal was to obtain quantitative information on the fetal DNA in maternal plasma. For this purpose we used real time quantitative PCR which allowed very accurate determination of DNA copy number. For fetal DNA quantitation, we used the SRY gene as a marker. A reference amplification signal generated using beta-globin specific primers and probe, was used to determine the total amount of fetal and maternal DNA. When applied to 40 pregnant women with gestational ages from 12 to 40 weeks, the proportion of fetal DNA was found to be between 0.45% and 8.3% during the second trimester, and between 4.3% and 11.6% during the third trimester. This very high enrichment of fetal DNA in maternal plasma is comparable to figures obtained using many state-of-the-art fetal cell enrichment protocols. These data therefore suggest that plasma DNA analysis may be an important new technology for prenatal screening and diagnosis.
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Pten gene mutations are seen in high grade, but not in low grade gliomas. Stenzel, T. T. 1, Rasheed, B. K. A. 1, McLendon, R. E. 1, Parsons, R. 2, Friedman, A. H. 3, Friedman, H. S. 4, and Bigner, S. H. 1 (1Depts. of Pathology, 3Surgery, and4Pediatrics, Duke Univ. Medical Ctr., Durham, NC; 2 Depts. of Pathology and Medicine, Col. Of Physicians & Surgeons, Columbia Univ., New York, NY).
The PTEN gene (l0q23) has been implicated as a tumor suppressor gene in brain, breast, and prostate tumors. Loss of heterozygosity for chromosome 10 alleles is seen in 60-90% of glioblastomas. In the present study, 123 brain tumors including various grades and histologic types of gliomas occurring in children and adults were analyzed for PTEN mutations by SSCP assay and sequencing. PTEN gene mutations were found in 13/42 adult glioblastomas and 3/13 adult anaplastic astrocytomas, while none of the 21 low grade adult gliomas nor any of the 22 childhood gliomas of all grades showed mutations. The single medulloblastoma (1/22) with a mutation, occurred in a recurrent tumor also possessing a p53 mutation. No mutation was detected in each single case of supratentorial primitive neuroectodermal tumor, ependymoblastoma and ganglioglioma. The mutations include deletions, insertions, premature stop codons, and missense mutations. A mutation hot spot was present in the tyrosine phosphatase region in exon 5 where mutations were found in 5 separate cases between codons 126 and 132. High grade adult gliomas with PTEN mutations included cases which contained gene amplification (EGFR, GLI, CDK4, PDGFA, or MDM2) or p53 gene mutations as well as cases which did not contain either abnormality. There was no obvious relationship between the presence of a PTEN mutation and survival, however, PTEN mutations tended to occur in older patients. We have not investigated in detail homozygous deletions of the PTEN gene in these tumors, and thus the observed frequency of PTEN gene alteration is likely underestimated. Regulatory region mutations as well as methylation represent additional possible mechanisms of inactivation. In conclusion, this analysis suggests that PTEN gene mutations are restricted to high grade adult gliomas.
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Homogeneous systems allowing integrated amplification and detection of point mutations. Whitcombe, D., Theaker, J., Gillard, H., Callaghan, K., Little, S. (Zeneca Diagnostics, Northwich, Cheshire, United Kingdom).
The ability to detect mutations in human genomic DNA is central to gene discovery and genetic analysis. There are many different PCR based methods available which will allow reliable detection of point mutations in a human DNA sample and the focus of research has now shifted to improving ease of use and throughput. Our approach has been the development of homogeneous PCR mutation analysis techniques. The key features of these methods are that all reagents are added to the reaction tube prior to PCR and that a signal is generated when the sample contains the mutation under analysis. The principle benefits are ease of use and minimisation of contamination. ARMS is a well established technique for the detection of point mutations in genomic DNA. ARMS acts as a molecular switch which converts the presence or absence of a point mutation into the presence or absence of a PCR product. We have used ARMS reactions to make amplification contingent upon the presence of specific mutations in a sample and then detected the presence or absence of the resulting PCR fragment. This simplified the task of the homogeneous analysis method from mutation detection to fragment detection. We have combined ARMS with amplicon specific detection to allow the simple analysis of point mutations. This method demonstrated the very high specificity of the ARMS molecular switch over the less discriminatory allele specific hybridization approach. The benefits included increased reliability, simplified probe design and the ability to detect point mutations in mixtures of mutant and normal DNA at ratios of 1:1000. The use of real-time detection further simplified test design and maximized the specificity of the method.
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Mapping of aflatoxin B1 DNA adducts in the human p53 gene by ligation-mediated PCR. Denissenko, M. F.1, Koudriakova, T. B.2, and Pfeifer, G. P.1 (1 Beckman Research Inst. of the City of Hope, Duarte, CA, 2 The Ohio State University, Columbus, OH).
Formation of DNA adducts at specific hotspot sites in the p53 gene is strongly implicated in genesis of certain types of human cancer. The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma diagnosed in regions with high food contamination by AFB1. To map AFB1 DNA adducts in human cells treated with the genotoxin we have used a ligation-mediated PCR (LMPCR) technique of damage detection at nucleotide resolution. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat or human liver microsomes. DNA adducts were released by hot piperidine treatment and LMPCR analysis performed using sets of primers specific for exon 7 (upper strand) of p53. Induction of adducts was detected at 40-400 µM concentrations of AFB1. Rat microsomal activation system generated AFB1 damage more efficiently than human system. Significant damage was seen at codon 249 (third base in the sequence AGG) which is a known mutational hotspot in liver cancers. Adducts were also formed at codons 245, 242, and 226. The data for the first time demonstrate site-selective formation of adducts in the human p53 gene upon cellular exposure to AFB1. The results show the usefulness of the LMPCR method for identification of carcinogen "fingerprints" in human genes pertinent to tumorigenesis. Supported by NIH grant ES03124.
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Universal ribonuclease resistant RNA standards (Armored RNATM) for RT-PCR and bDNA-based Hepatitis C virus RNA assays. DuBois, D. B.1, WalkerPeach, C.1, Pasloske, B. L.2, and Winkler, M.2 (1Centeron Diagnostics and 2 Ambion, Inc, Austin, TX).
Introduction: Development and standardization of amplification assays for HCV RNA has been hampered by the lack of universal controls for both comparing detection technologies and for evaluating the performance of individual laboratories. We have developed a new technology that allows the construction of bacteriophage protein-RNA complexes that incorporate predefined exogenous RNA sequences. These complexes are completely protected from plasma nucleases.
Methods/Results: A consensus sequence of the 5' NCR and core region HCV type 2b RNA was derived from 34 separate individuals and synthesized de novo using deoxyoligonucleotides and thermostable ligase. A 450 base dsDNA fragment was cloned into a proprietary protein expression/RNA packaging vector, and then expressed in E. coli. Resulting Armored RNATM (AR) constructs contain the complete, predefined RNA sequence, and maintain 100% sequence fidelity from design to packaged RNA as assessed by ABI dye-terminator analysis. Armored RNA is completely stable in human plasma at room temperature for extended periods of time, and is totally non-infectious. When spiked into seronegative, non-viremic plasma, these standards produce linear dilutions in both the Roche HCV MonitorTM assay, and in the Chiron QuantiplexTM HCV assay. Additionally, AR-HCV-2b standards produce highly reproducible signals and have other desirable performance characteristics in both assay formats.
Summary: We have extended Armored RNATM technology to include the construction of HCV genotype 2b RNA controls that function well in at least 2 leading assay formats. Additional applications for HCV diagnostics, such as HCV genotype-specific, and RNA sequencing standards are in development.
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Correlation of sample preparation methodology with efficiency of PCR. Loewy, Z. G.1,2, Dong, Q.2, Buffolino, P.3, Shaler, R.3, and Baum, H. J.3 (1 Orchid Biocomputer, Inc, Princeton, NJ, 2 Sarnoff Corp., Princeton, NJ, 3 Office of the Chief Medical Examiner, New York, NY).
Future advances in molecular diagnostics and genomics will require automation of sample preparation. The sample preparation chemistry will have to be fast and reproducible to ensure a reliable and robust automated system. In the present study, two different sample preparation methods were evaluated. The first method is the DNA DirectTM System by Dynal, Inc. In this approach, magnetic microparticles are used for affinity capture of DNA. The second method is a ChelexTM resin-based system. Blood samples boiled in the presence of ChelexTM result in purified DNA in solution. The relative efficiencies of the two methods were evaluated by comparing the PCR results of the ß globin locus. DNA obtained from 9 different blood samples isolated using both methods was amplified using two different enzymes, AmpliTaqTM and AmpliTaq GoldTM. Gel analysis of amplicon products demonstrated that the DNA DirectTM samples were cleaner as compared to ChelexTM samples. Quantitation of the amplicon products showed an apparent reduction in amplification efficiency with ChelexTM samples amplified with AmpliTaq GoldTM, but not with ChelexTM samples amplified with AmpliTaqTM. In contrast to the ChelexTM results, amplification efficiencies of DNA DirectTM samples were consistent when amplified with either of the two enzymes. The results suggest that the DNA DirectTM method is a fast and reliable method for the production of genomic DNA. The DNA DirectTM method is readily amenable to automation.
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Design, synthesis, and incorporation of photocleavable linkers into oligonucleotide probes for diagnostic applications. Xiang, G.1, Lough, D. M.2, Tang, K.1, and Köster, H.3 (1 Sequenom Inc., San Diego, CA, 2 Sequenom Insturments GmbH, Hamburg, Germany, 3 Department of Biochemisty and Molecular Biology, University of Hamburg, Hamburg, Germany).
Two photocleavable linkers have been designed, synthesized, and converted into their corresponding phosphoramidites for solid phase nucleic acid synthesis with high overall yield. Both phosphoramidites have comparable coupling efficiency with those of common phosphoramidite monomers. The photocleavable linker is placed between the sequencing primer and the spacer which is also conjugated to an amino group (or HS or Biotin group) for immobilization onto solid support. Preliminary results indicate that both photocleavable linkers can be cleaved completely within minutes upon irradiation by 365 nm UV light. Direct MALDI-TOFMS analysis without pre-irradiation also indicates partial photocleavage for the oligonucleotide conjugates containing photocleavable linker during MALDI processes.
By immobilization through the functional group (H2N, HS, or Biotin) on the 5'-end of the oligonucleotide conjugate (probe) containing photocleavable linker onto solid support the 3' end is available for biological reactions. The photocleavable linker should be stable to all conditions for the biological reactions. With suitable MALDI-TOFMS condition, direct analysis of the immobilized analytes on the solid support would be possible.
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Simplified heteroduplex analysis: a rapid method of detecting single-base substitutions in the low density lipoprotein receptor gene analysis. Ashavaid, T. F., Altaf, A. K., Rao, R., Nair, K.G., (Research Laboratories P. D. Hinduja National Hospital and Medical Research Centre, Mahim, Mumbai, India).
Familial hypercholesterolemia (FH) is a common autosomal dominant disease caused by numerous mutations in the low-density lipoprotein receptor (LDLR) gene. The mutational heterogeneity of the LDLR gene complicates disease diagnosis at the DNA level. Therefore, much interest is focused on screening methods. Heteroduplex analysis has become popular and useful in the fields of molecular investigation. The heteroduplex method currently in use is non-radioactive but time-consuming or requires the use of Hydrolink-MDE gels. A newly modified non-radioactive heteroduplex method is presented and applied to LDLR gene analysis for detecting mutations reported in Asians in exon 3,4,9 and 14. The method requires only small amounts of polymerase chain reaction (PCR)–amplified DNA and can readily resolve differences between mutant and normal DNA. DNA was amplified using same pair of specific primers. For heteroduplex analysis, very small aliquots of amplified products from both normal and patients are denatured and renatured in a programmed PCR thermal cycler. The denaturing/renaturing is a critical step which increases the chance of heteroduplex formation between non-homologous DNA strands. The renatured samples are loaded into a low cross-linking (1%C) 10% polyacrylamide gels containing 7.5% Urea in 1 x TBE buffer, electrophoresed at 200V for 3-5 h at RT, stained with ethidium bromide and observed under ultraviolet light. Using this method, we have detected three samples showing heteroduplex formation; two in exon 4 and one in exon 3. Sequencing of these samples are in process to detect the exact molecular change causing the heteroduplex formation. The efficacy of this technique was tested by using mutation-positive controls for exon 3 (R57C), exon 4(E207K), exon 9 (E384K) and exon 14 (P664L). It is not yet known, if all possible base pair change can be detected by the method described, but from the four known mutations that we have tested (relatively large PCR products, upto 500 bp) three (except exon 9, E384K, 550 bp) were detectable using this technique. This method is simple, rapid and gives clear and reliable results within short time. This approach can thus be the preferred initial method of screening for detecting known or unknown point mutations in the LDLR gene.
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Hyperhomocysteinemia and the prevalence of methylenetetrahydropholate reductase mutation in patients with coronary heart disease. Ashavaid, T. F., Nair, S., Li, S., Nair, K. G., Dalal, J. J., ( Research Laboratories, P.D. Hinduja National Hospital & Medical Research Centre, Mahim, Mumbai, India and Specialty Laboratories, California).
Hyperhomocysteinemia (HCA) has been identified as an independent risk factor for Coronary Heart Disease (CHD). HCA may be due to the impairment in the metabolism of methioninc by mutations in enzymes responsible for homocysteine metabolism. The alanine/valine (A/V) polymorphism of the 5, 10 methylenetetrahydrofolate reductase (MTHFR) gene, one of the key enzymes catalyzing remethylation of homocysteine, has been reported. This mutation results in a C to T substitution at nt 677 resulting in a chinge from alanine to valine. The MTHFR mutation correlates with increased plasma homocysteine levels as a result of the reduced activity and increased thermolabilty of this enzyme. Our study was aimed at examining the relationship between HCA and MTHFR status in patients with CHD. We have studied 115 patients with angiographically documented CHD and 115 controls referred to the P. D. Hinduja National Hospital, Mumbai, India. Total plasma homocysteine was estimated by HPLC using fluorescence detection and the MTHFR mutation was detected by PCR followed by digestion with Hmf 1 restriction enzyme. Thermolability for MTHFR mutation was determined from lymphocytes by radiochemical assay. The mean plasma homocysteine value was 14.52 nmole/ml (range 4.3-175.8, n=115) in patients as compared to 14.81 nmole/ml (range 4.1-82.7, n=115) in controls. The values were not found to be significantly different in both the groups. The heterozygous MTHFR mutation was found to be 54.54% and 14.28% in patients and controls with hyperhomocysteinemia respectively. The thermolabile MTHFR enzyme assay was found to correlate well with the PCR results. We conclude that the heterozygous MTHFR mutation correlates with elevated levels of plasma homocysteine in patients with CHD. The prevalence of heterozygous MTHFR mutation had been found to be 0.5% of the general population in Western studies. We have found the prevalence of heterozygous MTHFR mutation to be 4.34% in controls. There are no other reports from India on plasma homocysteine levels and MTHFR mutation. Since the HCA and MTHFR mutation are associated with cardiovascular disease, it would be worthwhile to screen patients for this potentially treatable risk factor.
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Advancements in methods for isolation of genomic DNA. Kuruc, M., Krupey, J., Mascoli, C., (LigoChem Inc., Fairfield, New Jersey).
ProCipitateTM is a patented, synthetically engineered polyelectrolyte which has unique properties in biological solutions. Its primary use is as a direct substitute for toxic solvents, particularly phenol and chloroform, in the isolation of DNA or RNA. The benefits of the described ProCipitateTM methods include versatility, yield and purity. In addition, the filtration characteristics of the protein-polymer complex accommodates high-throughput 96 well formats.
Procedures for isolating genomic DNA directly from whole blood are first presented using a modified buffy coat process followed by Guanidine Thiocyanate Iysis and ProCipitateTM addition. Typically 30-50 µgs per ml of genomic DNA is produced with A260/A280 ratios greater than 1.80. The DNA thus isolated is further demonstrated to be suitable for restriction endonuclease digestion and PCR.
A further enhancement utilizes a new solid-phase which precludes alcohol precipitation and thereby all centrifugation steps are eliminated in a 96 well filter format. The quality of DNA produced by this simple and fast process is demonstrated to be superior to other commercially available kits. It is also directly scaleable by volumetric ratio affording a great deal of economy and versatility.
It is foreseen that due to its non-volatile nature and superb protein extraction capacity, ProCipitateTM and associated products will be advantageous when DNA yield and purity are of paramount importance.
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Short tandem repeat typing for human identity testing using time-of-flight mass spectometry. Butler, J., Li, J., Shaler, T., Monforte, J., Becker, C., (GeneTrace Systems, Inc., Menlo Park, California).
DNA separations which traditionally have been performed by slab gel or capillary electrophoresis may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of allele sizing. The polymerase chain reaction (PCR) is used to amplify small amplicons, usually less than 100 bp in size, which bracket the repeat region in the STR markers. We are currently working with over 20 commonly used tetranucleotide repeats including HUMTH01, TPOX, CSF1PO, VWA, FES/FPS, F13A1, and the sex-typing marker amelogenin. PCR products under 100 bp in size may be analyzed in less than 10 seconds with single base resolution. With automated sample preparation on robotic workstations, thousands of genotypes may be obtained daily using a single mass spectrometer.
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A sensitive and reliable protocol for the PCR amplification and sequencing of the p53 gene from formalin-fixed, paraffin-embedded tissue sections. Cohen, M., Hasnah, H., Sallash, G., Leon, J., (Quest Diagnostics, San Juan Capistrano, California).
Alterations of the p53 protein can cause defective cellular responses to DNA damage. Detection of alterations in the p53 gene or protein is likely to be important for the management and treatment of cancers. DNA sequencing is a direct method to detect mutations of the p53 gene. Thus, the goal of our laboratory is to develop reliable and accurate protocols for sequencing of the p53 gene. Several good protocols for sequencing of the p53 gene from fresh tissue specimens and blood have been developed. However, fresh specimens are not always available, in which case, tissue samples in paraffin blocks may be used as an alternative. We have developed a sensitive protocol for extraction of genomic DNA from formalin-fixed, paraffin-embedded tissues. Serial sections of 5, 10, 20, 30, and 50 microns from lung, breast, and colon tissues were extracted. Xylene was used to dissolve the paraffin and a commercial tissue extraction kit with a final filtration step was used to extract and purify the DNA. Using this protocol, a sufficient quantity of high quality DNA which was sufficient for several PCR reactions was obtained from as little as a single 5-micron section. Amplification of exons 5 to 8 of the p53 gene was achieved from these DNA. PCR products were sequenced with T7 DNA Polymerase (Sequenase) using a solid phase DNA sequencing kit and fluorescently labeled extension products were detected in an automated DNA sequencer. High quality sequence data was obtained which allowed for easy detection of mutations including mutations present in mixtures of wild type sequence. We have developed a sensitive, reliable protocol for sequencing of the p53 gene from formalin-fixed, paraffin-embedded tissue. This protocol would be very useful for determining the p53 status in retrospective studies in which fresh tissue samples are not available.
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The incorporation of chemically-cleavable linkers into oligonucleotide probes for diagnostic applications. Lough, D.1, Klimscha, H.1, Xiang, G.2, Köster, H.2, (1Sequenom Instruments GmbH, Hamburg, Germany; 2 Sequenom Inc., San Diego, California).
A number of modifications are available allowing arrays of oligonucleotides to be constructed on a variety of surfaces. Sample work-up consists of a simple series of reagent exchange washes. Introducing a cleavable linker into the system allows the solid-bound DNA to be removed and subsequently analysed, eg by MALDI-TOF mass spectrometry.
The diisopropylsilyl linker is an attractive option for chemically-cleavable linkers. It can replace a phosphodiester linkage in DNA and so allow an intra-DNA cleavage site; it is cleaved rapidly in weakly acidic media; and it is compatible with final step of MALDI sample preparation: 3-hydroxypicolinic acid matrix. The linker may be pre-cleaved using a volatile acid (eg trifluoroacetic acid) before addition of the matrix, or the matrix may be doped with additional acid.
A comparison of DNA bound at the 5'-position, via an amido bond, and via the streptaviden-biotin interaction will be given with the cleavage products detected by MALDI. With this approach, redundant information is left behind on the solid support (hence lowering the mass of fragments detected). This strategy becomes particularly relevant for analysis of large DNA fragments, eg PCR products and Sanger sequencing ladders.
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Rapid quantitation of CMV infection by fluorescence detection of DNA amplification product. Sanders Sevall, J., Tirtorahardjo, B., (Specialty Laboratories, Santa Monica, California).
Detection of CMV-specific nucleic acids in clinical material by the polymerase chain reaction provides a rapid and highly specific diagnostic test for CMV infection. While in healthy CMV IgG-positive individuals PCR remains negative in the majority of cases, CMV DNA is readily detected in various specimens during acute infection with PCR, usually becoming positive prior to other diagnostic methods. However, when monitoring immunocompromised patients, unwanted positive results are often without further evidence of active CMV infection. To better discriminate between latent and active infections, a quantitative PCR offers a rapid and efficient method for CMV quantitation. In this report, the fluorescence detection of PCR amplification products allows a rapid and quantitative detection of CMV DNA between 10 and 1,000,000 copies, with a root mean square of the linear regression analysis of the standard curve of 0.999 and an intra-assay variation of less than 5%. A single tube extraction procedure and organic extraction method were compared for accuracy with the organic extraction procedure having an inter-run variation of 25%. In comparing two CMV quantitative amplification methods, there was good agreement between 31 specimens with a low positive specimen detected by the fluorescence detection procedure that was not detected by the CMV DNA detection kit. A rapid DNA amplification and in-tube detection of the amplicon by fluorescence means a rapid, sensitive and accurate DNA quantitation procedure.
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Cascade rolling circle amplification, a homogeneous fluorescence detection system for DNA diagnostics. Thomas, D.1, Lizardi, P.2, Nardone, G.1, Winn-Deen, E.1, (Oncor, Inc., Gaithersburg, Maryland1 and Yale University School of Medicine, New Haven, Connecticut2).
We have developed a homogeneous method of nucleic acid amplification and detection that is as fast, sensitive, and specific as existing technologies but improves upon them by being easier, more quantitative, and more accurate. Cascade rolling circle amplification (CRCA) is a novel isothermal amplification system that uses generic primers to amplify circularized DNA probes instead of the target. It is rapid and highly sensitive, consistently yielding over a billion-fold amplification within one hour without using a thermocycler. Carry-over contamination is minimized since the amplification and detection steps are performed in the same tube, without opening the tube after amplification. The amplification process begins by forming a circularized padlock probe resulting from ligation of the ends of a linear probe whose ends are both hybridized to adjacent target regions. This is followed by rolling circle amplification using two primers. By using a DNA polymerase with strong strand displacement activity, a cascade reaction occurs whereby displaced single-stranded tails are continually regenerated upon binding of primers and subsequent primer extension and strand displacement, forming a ladder of products from one to many unit lengths of the original circle. Homogeneous detection is achieved when one of the primers is an energy transfer labeled primer which generates a fluorescence signal only when incorporated into the amplified product. Using pUC19 as a model target, 90-base linear probes were ligated at a specific target site to form a padlock probe. Dilutions of the padlock were made and subjected to CRCA using Bst DNA polymerase (large fragment), one unlabeled primer, and one energy transfer labeled primer. Signals well above background were detected when using as few as 10 template circles during a 60-minute incubation at 64°C. Structures of both the labeled and unlabeled primers are specially designed to achieve strong signals without background artifacts. Energy transfer labeled primers contain a hairpin structure such that the donor fluorophore and acceptor moieties are sufficiently close for maximal quenching, but generate a significant fluorescence signal upon incorporation into the amplicon. Hairpins containing five- or six-base stems and similar size loops appear to be optimum in this system. The primer binding sites of each primer must be at least 18 bases to ensure efficient priming at 60-65°C, the preferred temperature range for the polymerase. Model systems for infectious diseases such as HIV and Chlamydia trachomatis are currently being tested to assess the clinical utility of this assay.
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Analysis of RNA transcripts using non-denaturing entangled polymer capillary electrophoresis. Sorensen, M., Holloway, J., Riggs, M., (Gen Probe, Inc., San Diego, California).
Utilizing a Gen-Probe T7 polymerase transcription system, RNA transcripts of the pol region of HIV (Human Immunodeficiency Virus) as well as the 5' UTR region of HCV (Human Hepatitis C Virus) were made from plasmid templates digested with 4 different restriction endonucleases to yield 4 distinct length RNA transcripts. These transcripts were used to create 4 member RNA ladders which were analyzed by capillary electrophoresis (CE) using a non-denaturing gel. Thus, the ladders yield a distinct slope or "fingerprint" when transcript size is plotted against migration time. As a result, native sequence HIV and HCV transcripts or closely related transcripts can be compared to their corresponding ladders to determine their nucleotide lengths. Moreover, this method can also distinguish transcripts of similar lengths based on their differing migration times caused by their unique conformations. Overall, CE is an excellent method for determining the quality of RNA transcripts. It does not require the time or the radioisotopes required by traditional northern blot methods and also features superior reproducibility (%RSD), resolution, sensitivity, and quantitiation.
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"Non-Infectious" RNA controls derived from the 8E5/LAV cell line. Howell, R., Manak, M., Wild, C., (Biotech Research Labs, Rockville, Maryland).
The 8E5/LAV cell line is a subclone of the LAV-infected A3.01 cell line (a HAT-sensitive CEM derivative) which contains a single integrated copy of proviral DNA coding for a defective HIV-1 particle. The defect to virus replication results from a frame shift mutation which obliterates RT activity. Other structural gene products are produced normally and assemble to form a retroviral particle. The 8E5/LAV clone was derived (Folks et al. J. Exp. Med. 164:280, 1986) from the A3.01 parent cell line infected with HIV-lLAV by repeated exposure to 5-iodo-2'-deoxyuridine (IUdR). The "non-infectious" nature of virus produced by the 8E5/LAV cell line makes it an attractive reagent for numerous applications. Recently, we produced several large volume, high titered stocks of this material for use in the preparation of standards and controls for HIV-1 clinical assays. For large scale virus production ~ 1.35 X 109 cells were seeded in 2.7 liters of tissue culture media. Virus was isolated from culture supernatants by centrifugation and purified by banding on a sucrose gradient. Samples from the original culture supernatants (1X), as well as material collected prior to (30X) and post-sucrose gradient purification (30X) were assayed for the presence of p24 antigen, RT activity, and virus infectivity. The resulting virus preparations contained high levels of p24 (15-5Ong/ml) and > 109 virus RNA copies/ml. Consistent with the original report no reverse transcriptase activity was detected in any of the samples. Quantitative infectivity experiments using A3.01 cell line targets showed no detectable infectivity in the original culture supernatants (1X), however, the concentrated material (30X) was found to contain infectious virus. Our results indicate that the titer of this material is at least 7 logs lower than that observed for the WT HIV-lIIIB virus and that viral RNA copy numbers of > 108 were necessary for successful infection. The virus is very stable in plasma and works well with current HIV RNA assays. The 8E5/LAV virus is a convenient "non-infectious" source of HIV- 1 RNA for use as RNA controls and standards.
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Advances in kinetic thermal cycling technology. Watson, R., Higuchi, R., Laird, W., Saiki, R., (Roche Molecular Systems, Alameda, California).
The kinetic thermal cycler (KTC) system is a convenient, quantitative, and homogenous system for the real time monitoring of PCR amplification reactions. A fluorescent indicator dye that preferentially binds double-stranded DNA is used to signal the formation of amplification product. This sequence independent signaling technology depends on highly specific amplification reactions and requires the suppression of both mispriming events and "primer dimer" artifacts. We describe reaction modifications and primer design strategies that increase reaction specificity and allow for the reliable detection of low copy number genomic DNA targets. The linear relationship between signal intensity and the size of the amplification product is shown over a range of molecular weights. Under primer limiting conditions, the signal plateau that is observed is a function of the size of the product formed and can be used to distinguish larger, intended products from smaller primer artifacts. The use of a kinetically monitored melting step at the end of amplification further identifies the nature of the product formed and clearly differentiates between intended product and primer artifacts. Kinetic PCRs of multiple target sequences are monitored as multiplex reactions and the specific amplicons identified by signal plateau values and melting behavior.
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Highly sensitive detection of HIV type O and type M variants with transcription mediated amplification. Wang, R., Yang, Y., Dockter, J., Riggs, M., McDonough, S., Mimms, L., Giachetti, C., (Gen-Probe Incorporated, San Diego, California).
Assessment of HIV-1 RNA in plasma is a useful marker for viral infection. HIV-1 RNA appears in plasma prior to antibody response, providing a very sensitive method for early detection of HIV infection. A major challenge for the development of screening assays for this virus is its high degree of genetic diversity, which includes two major groups M and O. and several subtypes or Lades: A to I. With the goal of providing a rapid assay for routine analysis of large numbers of samples, we have applied Transcription Mediated Amplification (TMA) and the Hybridization Protection Assay (HPA) to HIV-1 RNA detection.
The sensitivity of the assay is at least 200 copies/ml of plasma spiked with HIV-1 IIIB virions. The sensitivity of detection of the different subtypes was evaluated using cell-free transcripts, viral isolates, and specimens representing the different viral variants. Our results indicated that the sensitivity of the HIV assay is similar for all eight subtypes tested: A to H. Moreover, testing of 33 type O viral isolates and specimens from 16 patients infected with type O virus indicated in all the cases very good detection.
In summary, the use of TMA and HPA technologies resulted in a reliable and highly sensitive assay for detection of all known HIV-1 subtypes. The development of this simple assay will have a measurable impact on the safety of the blood supply.
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A novel homogeneous fluorescence detection method for DNA amplification. Winn-Denn, E., Nazarenko, I., Nardone, G., Uehara, H., Hohman, B., (Oncor Inc., Gaithersburg, MD).
Simplification is a driving force in the implementation of DNA-amplification based assays in the clinical laboratory. In addition, practical use of this type of assay requires control of laboratory contamination from the amplicon generated during each assay. We have developed a homogeneous detection method which satisfies both of these needs.[Nazarenko et al., Nucl. Acids Res. 25, 2516 (1997)]
The SunriseTM amplification detection system is based on the incorporation of energy transfer-labeled primers directly into the PCR amplicon, and the subsequent generation of fluorescence. The Sunrise primer contains a target specific sequence on its 3' end, a hairpin structure on its 5' end, and a fluorescence donor-quencher pair which are located in close proximity on opposite strands of the hairpin stem. The hairpin structure brings the fluorophore and the quencher together when the primer is free in solution, providing efficient quenching and low fluorescence background. A signal to noise ratio of 35:1 was obtained using hairpin primers labeled with fluorescein and a non-fluorescent quencher, 4-(4'-dimethyla minophenyazo)benzoic acid (DABCYL). When the primer is incorporated into the PCR amplicon, the hairpin structure is disrupted and a fluorescent signal is generated. This signal can be quantitated in real time using sealed tubes on thermal cyclers capable of monitoring fluorescence during PCR (PE Applied Biosystems PRISM 7700, Idaho Technology Lightcycler), measured at endpoint using a fluorescent microtitreplate reader or a spectrofluorimeter, or observed visually on a UV transilluminator. The utility of SunriseTM primers has been demonstrated using PCR for C. trachomatis, RT-PCR for prostate specific antigen cDNA, in situ PCR of lymph node tissue for HIV infection, methylation-specific PCR for p16, TRAP-PCR for telomerase activity, and allele-specific PCR for beta-3 androgen receptor mutations.
In addition to such target specific systems, a universal SunriseTM primer has been developed which can be easily adapted to any existing PCR assay system by simply resynthesizing one or both of the existing PCR primers with a universal tail sequence. The utility of the universal design has been shown using assays for IL-2 and p16 from human genomic DNA, and using C. trachomatis and HIV-1 infectious disease targets.
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Adapting the Gen-Probe rRNA-amplified mycobacterium tuberculosis direct test to the automated VIDAS instrument. Clark-Dickey, K.1, Hasselkus-Light, C.1, Knott, C.1, Longiaru, M.1, Nelson, N.1, Quigley, T.1, Santa Ana, S.1, Weinbaum, B.1, Catanazariti, L.2, Kluttz, B.2, McKinley, G.2, Moe, J.2, Vera-Garcia, M.2, (1Gen-Probe, San Diego, California and 2bioMérieux Vitek, Rockland, Massachusetts).
Tuberculosis remains a major global public health issue. Clinical laboratories desire fast and simple infectious disease diagnosis, which requires automation of state-of-the-art technology. Gen-Probe has introduced an isothermal Transcription-Mediated Amplification (TMA) that allows for the identification of M. tuberculosis (M.tb) in less than one day. bioMérieux Vitek's automated immunoassay instrument, VIDAS, uses an architecture that allows for versatile testing of samples. The coupling of TMA technology and the VIDAS enzyme-linked fluorescence detection would permit the clinician to identify infection in patients with less hands-on time and more ease than conventional methods.
Amplification of a respiratory specimen's rRNA occurs during the TMA reaction. The amplified product is then hybridized to a specific probe and detected on the VIDAS instrument. This method demonstrated complete specificity for members of the M.tb complex and did not cross react with a panel of 14 species of mycobacteria other than M.tb or 8 related genera. The sensitivity of the system is 1 fg of rRNA, or less than one M.tb cell per test. Ninety-eight clinical specimens were tested and the results compared to culture. Six M.tb culture positive specimens were positive by the amplified method. Three additional specimens were positive by amplified testing but culture negative; these were all resolved to be from patients undergoing treatment for pulmonary tuberculosis. There was no cross reaction with specimens identified by culture to contain M. gordonae, M. fortuitum, or M. avium. Eighty-nine specimens were negative for M.tb by both methods. In addition, bloody specimens did not elevate background levels. The union of probe diagnostic technology with an existing automated instrument functioning in clinical laboratories worldwide will allow for rapid and convenient identification of M. tuberculosis.
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A novel quantative assay for telomerase activity. Joe, B., Chang, S., (Roche Molecular Systems, Alameda, California).
Telomeres, 6-nucleotide repeats at the chromosome ends, shorten progressively with each cell division in normal somatic cells. Telomerase is a ribonucleoprotein that maintains the length of the telomere in germline and stem cells via reverse transcription. Telomerase activity has been observed in 80-90% of tumors of various types of cancer. The reactivation of telomerase had been proposed to be critical to the immortality of tumor cells. A PCR-based telomerase activity assay (TRAP) was first developed by Kim et al.
R. Higuchi et al developed a simple, quantitative assay to monitor the increase in fluorescence of ethidium bromide that results from its bonding to double-stranded PCR products during amplification. We have converted the gel-based TRAP assay to a kinetic PCR format. An advantage of the adapted assay is the opportunity to generate complete quantitative data in less than 3 hours after cell preparation. The 293 and WIN cell lines, respectively, were used as telomerase positive and negative controls. This kinetic TRAP assay has single cell sensitivity and a four-log dynamic range, and is able to detect 10 telomerase positive cells in the presence of 10,000 telomerase negative cells. The technical challenges that remain include the development of a simpler, more robust procedure for cell lysis and improved stability of the resulting telomerase activity. The contribution of telomerase activity from non-oncogenic cells such as in peripheral blood will need to be resolved. This simple, sensitive, non-radioactive, high throughput format has the potential to be employed for screening by large numbers of clinical specimens, e.g. urine and find needle aspirate for bladder and breast cancer respectively. Reaction optimization will be discusse
