Single Primer Polymerase Chain Reaction Research Articles

Characterization and application of a quantitative DNA marker that discriminates sex in chinook salmon (Oncorhynchus tshawytscha)

A qualitative male-specific DNA marker (OT-24) was amplified by spPCR (single-primer polymerase chain reaction) from chinook salmon (Oncorhynchus tshawytscha ) DNA along with several non-sex-linked products. The termini of the male-specific product were sequenced, and a pair of PCR primers were constructed for marker-specific PCR amplification. Dual primer PCR (dpPCR), with the marker-specific primers, amplified a product from both males and females. The amount of dpPCR product amplified from males was at least 100-fold greater than that from females. The quantitative difference between males and females was consistent among geographically distinct populations from western U.S. rivers. In addition, DNA sequence analysis indicated that OT-24 was highly conserved among geographically distinct salmon populations. The qualitative spPCR product segregated through several genetic crosses indicating equal sex ratios among progeny. Identification of the male and female juveniles by dpPCR was consistent with the spPCR analysis. There was no tissue specificity observed by spPCR or dpPCR analysis of this marker. A rapid DNA extraction method and dpPCR analysis were used to nonlethally determine sex ratios in wild spring chinook salmon adults, withheld for genetic and behavioral studies, prior to their development of gross sexual differences in their external morphology. Resume : Nous avons amplifie un marqueur qualitatif de l'ADN specifique aux mâles (OT-24) par spPCR (reaction en chaine de la polymerase a amorce unique) a partir de l'ADN de saumon quinnat (Oncorhynchus tshawytscha ), ainsi que plusieurs produits non lies au sexe. Les extremites des produits specifiques aux mâles ont ete sequencees, et une paire d'amorces de la

Non-isotopic PAGE analysis of amplified products from simple sequence repeat single-primer polymerase chain reaction

We describe modifications to two genetic typing procedures, simple sequence repeat (SSR)-anchored polymerase chain reaction (PCR) and single primer amplification of SSRs (SPARs), by combining polyacrylamide gel (PAGE) resolution of PCR amplified products with silver staining for detection. Turkey (Meleagris gallopavo) and chicken (Gallus domesticus) genomic DNA were used as templates in the PCR typing. The single primers used for PCR analyses were (CAC), (TCC), (GACT), (TGTC) and (TTTA). The PCR conditions have previously been described. As expected, the number of fragments detected by the analyses were higher than those previously described using agarose but lower than that reported by others from PAGE analyses and radioisotope detection. Unlike agarose gel analyses, all the primers amplified polymorphic products ranging from 22 percent (%) for (TGTC) to 44% for (TCC) (Table 1). The results suggest that the level of polymorphic DNA fragments from genetic typing by SPARs of SSRs could be increased, above that of agarose and ethidium bromide staining, by PAGE analyses followed by silver staining for detection. The level of DNA polymorphism detected was however lower than radioisotope detection, but the safety and ease of the modified method described in the current work may make it a preferable approach to both SPARs and SSR-anchored PCR for genetic mapping in eukaryotes.

Direct detection of expanded trinucleotide repeats using PCR and DNA hybridization techniques.

Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

ABSTRACTThe recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)‐ and (G + C)‐rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)‐rich primers was positive (i.e. band‐producing). Of the (G + C)‐rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair‐group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out‐group for the UPGMA analysis, showed a high degree of dissimilarity.

Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles.

The use of additional primers in the standard two primer polymerase chain reaction (PCR) is described. This modification allows detection of a target gene in a single reaction, and identification of the amplification product obtained or recognition of a specific allele. The oligonucleotides used are internal to the original amplification primers and amplification-compatible with one of the original primers. Annealing of an additional primer to the target gene as well as to the primary amplification product will lead to the appearance of an additional smaller amplification fragment upon agarose gel electrophoresis of PCR products. Use of one or more allele-specific oligonucleotides as additional primers, in addition to two gene-specific primers, will allow recognition of different alleles of the target gene in a single PCR without further analysis except gel electrophoresis. The general applicability of the method was determined with several PCR assays for the detection of pathogenic bacteria.

Multilocus markers for mouse genome analysis: PCR amplification based on single primers of arbitrary nucleotide sequence.

Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg2+, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.