PCR methods and applications最新文献

Random amplified polymorphic DNA (RAPD) markers offer quick screening of different regions of the genome for genetic polymorphisms. The standard RAPD procedure uses a single 10-base-long random oligonucleotide as a primer to amplify short stretches of the genome by PCR. We modified the procedure by using two primers in each reaction in a Brassica napus mapping project. We found that the two-primer RAPD tends to amplify more and smaller fragments than the standard RAPD technique. These new bands were always amplified in the two-primer reactions, and Southern analysis revealed that they had no homology to the bands amplified in single-primer reactions involving the same primers. Furthermore, these new markers were not linked to markers amplified with the same primers in the standard RAPD reactions, suggesting that they were amplified from different genomic regions. The advantage of the two-primer RAPDs is that it allows more reactions to be carried out with a limited number of primers to generate more markers. Using a single primer, the number of reactions is equal to the number of primers (n), which in turn limits the total number of markers. When using two primers in all possible combinations, the total number of reactions increases to n x (n-1/2). This method could be useful in conjunction with bulked segregant analysis to develop high density maps of certain chromosomal regions. We used this approach to map a second marker linked to a gene governing low linolenic acid concentration in a B. napus F2 population.

We have developed an efficient method for site-directed mutagenesis using two subsequential rounds of PCR. In this method, PCR conditions are optimized to favor high fidelity of Taq DNA polymerase in the presence of equimolar concentrations of MgCI2 and dNTP in the reaction mixture (pH 5.5-6.2). This method makes use of a pair of universal primers and the multiple cloning site of pUC/M13 vectors. Only one mutagenic primer is required per target site. In the second round of PCR, the 3' extension of the wild-type DNA strand is blocked by the presence of a segment of nonhomologous sequence at its 3' end, and as a consequence, the amplified, full-length DNA fragment is chiefly from the mutant strand. Furthermore, because the mutated DNA fragment has flanking restriction sites different from those of the wild-type DNA fragment, the wild-type DNA fragment is totally excluded in the step involving selective cloning of the mutant DNA fragment. This method was successfully used to introduce four, nonadjacent mutations in the 5' regulatory region of the cytochrome P450BM-3 gene. All 20 analyzed clones from these four cases of mutagenesis carried the desired mutations, and no undesired mutations were observed. We observed that the larger the number of mismatched nucleotide residues in the mutagenic primer, the higher the concentration of MgCI2 was necessary for successful PCR amplification. Our experimental results indicate that this method offers improvements in efficiency, flexibility, and fidelity.

Methods for identifying isolates of various pathogenic bacteria by DNA fingerprinting with random primers (RAPD) have been described recently. In these methods many primers are screened and the primers that generate the most informative DNA pattern are selected. A new strategy that simplifies the primer selection process for RAPD fingerprinting has been developed in our laboratory. In this approach, one or more degenerate nucleotides is introduced into the core RAPD primer sequence at various nucleotide positions. Results show that a single degenerate nucleotide in the primer sequence can significantly change the DNA profile obtained for the same template. The more removed the degenerate nucleotide is from the 3' end of the primer, the less dramatic is its effect on banding pattern. This method utilizing degenerate RAPD (D-RAPD) primers was tested on clinical isolates of Legionella pneumoniae, and results were confirmed with nondegenerate RAPD primers. Results obtained with D-RAPD primers were in total agreement with those obtained with nondegenerate RAPD primers. We propose that the use of a core RAPD primer sequence with one or more degenerate nucleotide(s) at various positions can expedite the generation of unique DNA fingerprints individual organisms. A general method for selecting the most useful fingerprinting RAPD primers is discussed.

Single PCR primers complementary to microsatellite repeats were used to amplify genomic DNA samples from various plant species, as well as from human, yeast, and Escherichia coli DNA. Most primers generated distinct amplification products, resulting in fingerprint-like banding patterns after agarose gel electrophoresis and ethidium bromide staining. These fingerprints allowed distinction among different plant taxa at an interspecific as well as intraspecific level. Unexpectedly, some of the primers produced bands with the E. coli template DNA as well. A detailed examination of the influence of PCR conditions, especially the annealing temperature, on the quality of banding patterns suggested that the majority of bands were generated by mismatch priming in a way similar to random amplified polymorphic DNAs (RAPDs).

Multiple fluorescence-based PCR single-strand conformation polymorphism (MF-PCR-SSCP) with postlabeling was developed. The target sequence was amplified by PCR using unlabeled primers. Free dNTPs were removed from the amplified products by ethanol precipitation. The dNTPs at the 3' ends of the amplified DNA fragments were exchanged with fluorescent dUTPs or ddNTPs using Klenow fragment of DNA polymerase I. The DNA fragments labeled with fluorescent dUTPs or ddNTPs were heat denatured and applied to a nondenaturing polyacrylamide gel set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by the computer program Genescan 672. By use of MF-PCR-SSCP with postlabeling, seven different single base mutations of the human K-ras oncogene were detected even under one electrophoresis condition.

We describe a multiplex PCR assay for the detection of bcr-abl fusion mRNA in Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL). The assay provides a quick method for screening p190 (e1:a2) and p210 (b2:a2 or b3:a2) bcr-abl mRNAs simultaneously. The assay proves to be highly sensitive with detection of as little as one positive bcr-abl-expressing cell in a background of 10(5) negative bcr-abl cells. Bone marrow and peripheral blood specimens from six patients were in total accordance when run by multiplex PCR and by the single primer PCR approach. The multiplex bcr-abl assay may prove to be highly useful for screening newly diagnosed patients with ALL for the bcr-abl fusion transcript and in following the course of disease during therapy.

We have developed a new procedure (Vir typing) for typing Streptococcus pyogenes, by amplifying the entire 5- to 7-kb variable vir regulon by long PCR. The amplified DNA is then cleaved with HaeIII and visualized by ethidium bromide fluorescence after agarose gel electrophoresis. A simple procedure for preparing DNA of sufficiently high quality from 96 samples was employed simultaneously. This DNA was also used to develop a random amplified polymorphic DNA (RAPD) procedure. The discriminatory power of the two DNA-based procedures was compared with previous methods, M typing, and multilocus enzyme electrophoresis. Both procedures were highly discriminatory, but the stoichiometric yield of restriction fragments in Vir typing allows unambiguous interpretation of results.