PCR methods and applications最新文献

We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.

A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed that allows for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method that combines in one assay DNA amplification by PCR with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I, target DNA is amplified using high-melting primers (Tm values between 68 degrees C and 89 degrees C) in a two-step PCR cycle that employs a 94 degrees C denaturation step and a 72 degrees C anneal-elongation step. In stage II, genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes (Tm values between 51 degrees C and 67 degrees C) located between the PCR primers is accomplished by several cycles of denaturation at 94 degrees C followed by anneal-ligation at 55 degrees C. Ligation products are fluorochrome-labeled at their 3' ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used successfully to analyze human genomic DNA for cystic fibrosis (CF) alleles. Because product detection occurs concurrently with target amplification, the technique is rapid, highly sensitive, and specific and requires minimal sample processing.

The 5' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase in reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-->3' nucleolytic activity of Taq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotide. In all cases, the reporter dye was attached to the 5' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes. It is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.

A rapid and sensitive technique is described that measures the length of the poly(A) tail on a specific mRNA within subnanogram quantities of total cellular RNA [the Poly(A) test (PAT)]. In a single-tube reaction, a poly(dT) primer is synthesized in situ on the poly(A) tail of mRNAs using oligo(dT) and DNA ligase. By modulating the annealing temperature and primer concentrations, a GC-rich adapter sequence is targeted to the 5' end of the poly(dT) primer. This ligated poly(dT)-anchor is then used to prime reverse transcription of the mRNA, yielding a library of PAT cDNAs. The length of a poly(A) tail is determined by PCR amplification using the oligo(dT)-anchor primer and a message-specific primer. Comparison of PCR products from different samples allows quantitative determination of changes in polyadenylation of a given mRNA. This technique overcomes many of the pitfalls associated with conventional poly(A) tail length assessments and should prove useful in studying a variety of processes relating to polyadenylation.

We have developed a rapid, nonradioactive screening test enabling the simultaneous analysis of three low-density lipoprotein receptor (LDLR) gene mutations (D154N, D206E, and V408M), which together account for familial hypercholesterolemia (FH) in approximately 90% of the South African Afrikaner population. The assay is designed so that FH patients, negative for these founder-related mutations (found in descendants of European settlers), subsequently can be screened for unknown mutations in the mutation-rich exon 4 of the LDLR gene. Our screening assay consists of two steps: (1) multiplex allele-specific PCR amplification of exons 4 and 9, and (2) simultaneous analysis of single- and double-strand conformational polymorphisms in exon 4 by vertical electrophoresis on low cross-linked polyacrylamide gels. The simplicity, specificity, and versatility of the multiplex assay makes it an ideal system for routine screening of FH mutations in large population samples.

We describe the use of the quantitative PCR (QPCR) system 5000 (Perkin-Elmer) and competitive PCR in a simple and reproducible assay format for use in establishing a screen for the discovery of compounds that affect gene regulation. Insulin-like growth factor 1 (IGF-1) mRNA was chosen as an initial target to test the sensitivity and reproducibility of the QPCR System 5000 in the quantitation of PCR products generated in competitive PCR reactions. We found that with the use of sequence-specific probes, the QPCR 5000 could be used easily to distinguish between internal standard (IS) and wild-type products in PCR reactions. We were able to detect as little as twofold changes in cDNA amounts by using dilutions of total rat liver cDNA as a source of IGF-1 message. The QPCR system 5000 could be used to analyze 24 competitive PCR reactions (48 samples), single determinations, in approximately 1 hr. The flexibility, automation, and sensitivity of the QPCR System 5000 makes it a useful tool to measure the transcriptional regulation of various mRNAs.

Reverse transcriptase-polymerase chain reaction (RT--PCR) has been widely utilized for both the qualitative and quantitative assessment of the levels of specific mRNA transcripts in living systems. Quantitation of specific transcripts has often proved to be problematic because of the difficulty associated with relating the PCR-amplified product to the starting cDNA representing the mRNA of interest. We have overcome these difficulties and have developed a competitive PCR assay employing the property of electrochemiluminescence for the detection of PCR products. This assay possesses the dual advantage of being both nonradioactive and highly sensitive.

Polymorphic microsatellite markers are widely used in molecular analyses. The range of allele sizes and the allele frequencies within a population are important characteristics of the marker. Their determination previously has involved genotyping a large number of individuals. We have developed a technique for defining these characteristics by coamplification of many samples in a DNA pool. Groups of 32 and 42 DNA samples were genotyped and results were compared with those from individual genotype determinations. To improve the accuracy in the estimation of allele frequencies, arithmetic removal of stutter bands was carried out and the consistency of each marker was characterized. This approach was also applied to a group of 94 individuals. All of the work has been done using nonradioactive methods. Potential applications of this technique are in population genetics, high throughput genotyping, and loss of heterozygosity studies.