PCR methods and applications最新文献

The ligation-independent cloning of PCR products (LIC-PCR) is a versatile and highly efficient cloning procedure resulting in recombinant clones only. Recombinants are generated between PCR products and a PCR-amplified vector through defined complementary single-stranded (ss) ends artificially generated with T4 DNA polymerase. This procedure does not require restriction enzymes, alkaline phosphatase, or DNA ligase. The primers used for amplification contain an additional 12-nucleotide sequence at their 5' ends that is complementary in the vector- and insert-specific primers. The (3'-->5') exonuclease activity of T4 DNA polymerase is used in combination with a predetermined dNTP (dGTP for the inserts and dCTP for the vector) to specifically remove 12 nucleotides from each 3' end of the PCR fragments. Because of the complementarity of the ends that are generated, circularization can occur between vector and insert. The recombinant molecules do not require in vitro ligation for efficient bacterial transformation. To make this technique widely applicable, we have simplified the handling of the PCR fragments prior to LIC. The PCR products do not need further purification following the T4 DNA polymerase treatment. Incubation of vector and insert PCR fragments for as little as 5 min is sufficient for a high yield of recombinants. Comparison of the transformation efficiencies using different-length LIC tails revealed that using 12-nucleotide cohesive ends produced four times more transformants than were obtained with the LIC with 10-nucleotide cohesive ends. When the LIC tails were 8 nucleotides long, no transformants were obtained. PCR fragment purification, T4 DNA polymerase treatment, and LIC is complete in < 1 hr.

A differential PCR-based assay is presented that increases the accuracy of quantification of C-erbB-2 gene-copy number in DNA extracted from archival tumors. The C-erbB-2 gene is amplified in a high percentage of human adenocarcinomas arising at numerous sites, including breast, lung, and stomach. A number of studies have correlated C-erbB-2 with poor prognosis. Gene copy number may be relevant in identifying patients with different clinical outcomes. In this study a target gene and a single copy reference gene were coamplified in the same reaction tube. The level of target gene amplification was reflected by the ratio of the two resulting PCR products. Cell lines exhibiting variable copies ranging from 1 to > 8 of the C-erbB-2 gene were used as quality controls. This technique can reliably show a single copy difference between cell lines and can be used to semiquantitatively estimate gene copy number in DNA extracted from archival paraffin-embedded samples.

A major problem in the application of PCR is contamination with material amplified previously. Repeated PCRs result in the accumulation of intact and degraded amplicons and primer artifacts that can contaminate following amplification reactions. Post-PCR UV treatment and pre-PCR uracil DNA glycosylase (UDG) digestion have been recognized to efficiently inactivate or decompose intact amplification fragments. We show here that degraded amplification products and primer artifacts account for decreased sensitivity and may cause false-negative results. Our experiments indicate that partly degraded PCR products and primer artifacts containing sequences homologous to the primer oligonucleotides in the succeeding PCR reaction compete efficiently with sample DNA for the primers. The experiments done in this study may explain unexpectedly low PCR sensitivities reported in an increasing number of publications. In an attempt to solve this problem, we evaluated three post-PCR treatment methods to completely eliminate sequences competing for the amplification primers, namely, 8-methoxypsoralen (MOPS) or hydroxylamine treatment of amplified DNA and use of oligonucleotides containing 5'-ChemiClamps. However, all three methods did not sufficiently inhibit artificially produced carryover contaminations. In conclusion, false-positive results can be eliminated with UDG or UV treatment, but physical barriers are indispensable to avoid the occurrence of false-negative results.

The goals of the present experiments are (1) to improve dideoxy fingerprinting (ddF) and (2) to utilize ddF as a tool to evaluate the relative merits of different conditions for single-strand conformation polymorphism (SSCP). ddF is performed by electrophoresing one dideoxy termination reaction through a nondenaturing gel. The ddF pattern can be divided into a "dideoxy component" and an "SSCP component". If dideoxy CTP (ddCTP) is utilized for the termination reaction of ddF, the dideoxy component is abnormal when an extra segment is produced by a sequence change that creates an extra C or when a segment is eliminated by a change of C to another base. All subsequent segments produced by the termination reaction constitute the SSCP component that contains the mutation in a nested series of ddCTP termination products. The SSCP component is informative if abnormal mobility is detected in one or more of the segments. Herein, we utilize 84 different single-base changes in the human factor IX gene to examine the effects of gel matrix, temperature, and different primers on the sensitivity of ddF. The effects of glycerol and cross-linker ratio were examined on fewer mutations. The following conclusions emerge: 1. The sensitivity of the dideoxy component is invariant, but the sensitivity of the SSCP component can vary greatly with gel matrix, temperature, segment size, and sequence context. 2. For a given segment containing a mutation, it is likely that a mobility shift will be seen under some conditions but not under other conditions. By examining the mobility of the SSCP component in > 2200 segments, it was found that some conditions are statistically more likely to result in altered mobility, thereby increasing the average sensitivity of mutation detection by ddF or conventional SSCP. 3. GeneAmp and MDE gels are superior to polyacrylamide gels and electrophoresis at 8 degrees C is superior to electrophoresis at 23 degrees C. GeneAmp at 8 degrees C provided the highest SSCP component efficiency of all conditions tested; all 84 hemizygotes and 40 heterozygotes were detected readily by ddF under these conditions. 4. The segments that terminate near the mutation site are likely to show an abnormal mobility on polyacrylamide gels at 23 degrees C. 5. The likelihood of mobility shifts decreases with segment size, but sequence context can have a major effect on SSCP component efficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or = 3 of the 4 nucleotides in the cDNA step and the gLCR step. Fewer than 50 copies of synthetic RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.