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Selection for 3'-end triplets for polymerase chain reaction primers.

ABSTRACT: Primer extension by thermostable DNA polymerase in PCR starts from the 3'-end of a primer. If the PCR starting process fails, the entire PCR fails. Primer sequences at the 3'-end often interfere with success in PCR experiments. Over 2000 primer sequences from successful PCR experiments used with varieties of templates and conditions were analyzed for finding frequencies of the 3'-end triplets. This chapter discusses a trend in 3'-end triplet frequencies in primers used in successful PCR experiments and proposes requirements for the 3'-end of a primer. Finally, a method to select primers with the best 3'-end triplets is introduced based on the 3'-end analysis result.

SUBMITTER: Onodera K

PROVIDER: S-EPMC7121268 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Selection for 3'-end triplets for polymerase chain reaction primers.

Onodera Kenji K

Methods in molecular biology (Clifton, N.J.) 20070101

Primer extension by thermostable DNA polymerase in PCR starts from the 3'-end of a primer. If the PCR starting process fails, the entire PCR fails. Primer sequences at the 3'-end often interfere with success in PCR experiments. Over 2000 primer sequences from successful PCR experiments used with varieties of templates and conditions were analyzed for finding frequencies of the 3'-end triplets. This chapter discusses a trend in 3'-end triplet frequencies in primers used in successful PCR experime ...[more]

PMID: 17951790

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Project description:BackgroundChoosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range.ResultsWe present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers.ConclusionsWe describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.

Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

Selection for 3'-end triplets for polymerase chain reaction primers.

Publications

Selection for 3'-end triplets for polymerase chain reaction primers.

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