J Vet Sci.  2017 Dec;18(4):465-470. 10.4142/jvs.2017.18.4.465.

Comparison of allele-specific PCR, created restriction-site PCR, and PCR with primer-introduced restriction analysis methods used for screening complex vertebral malformation carriers in Holstein cattle

• Avanus K
• Altınel A

• KMID: 2398505
• DOI: http://doi.org/10.4142/jvs.2017.18.4.465

Abstract

Complex vertebral malformation (CVM) is an inherited, autosomal recessive disorder of Holstein cattle. The aim of this study was to compare sensitivity, specificity, positive and negative predictive values, accuracy, and rapidity of allele-specific polymerase chain reaction (AS-PCR), created restriction-site PCR (CRS-PCR), and PCR with primer-introduced restriction analysis (PCR-PIRA), three methods used in identification of CVM carriers in a Holstein cattle population. In order to screen for the G>T mutation in the solute carrier family 35 member A3 (SLC35A3) gene, DNA sequencing as the gold standard method was used. The prevalence of carriers and the mutant allele frequency were 3.2% and 0.016, respectively, among Holstein cattle in the Thrace region of Turkey. Among the three methods, the fastest but least accurate was AS-PCR. Although the rapidity of CRS-PCR and PCR-PIRA were nearly equal, the accuracy of PCR-PIRA was higher than that of CRS-PCR. Therefore, among the three methods, PCR-PIRA appears to be the most efficacious for screening of mutant alleles when identifying CVM carriers in a Holstein cattle population.

Keyword

Holstein cattle; Turkey; carrier; complex vertebral malformation

MeSH Terms

Alleles
Animals
Cattle*
Gene Frequency
Humans
Mass Screening*
Methods*
Polymerase Chain Reaction*
Prevalence
Sensitivity and Specificity
Sequence Analysis, DNA
Turkey

Figure

• 

  Fig. 1 The images of sequence allignment after DNA sequencing (A) and agarose gels from electrophoresis performed after allele-specific polymerase chain reaction (AS-PCR; B), created restriction-site PCR (CRS-PCR; C), and PCR with primer-introduced restriction analysis (PCR-PIRA; D), respectively. In panels B–D, images in lanes 1 to 4 belong to the same samples (w/w, non-carrier; w/m, carrier). (A) Genotypes of mutant and wild-type alleles that were aligned with the reference sequence of the bovine SLC35A3 gene (HM183012.1). (B) Image after AS-PCR amplification of SLC35A3 gene on 2% agarose gel. L, ladder; Lanes 1–3, carrier cattle (395 bp); Lane 4, non-carrier. (C) Image of CRS-PCR amplicons on 3% agarose gel after digesting with RsaI enzyme. L, ladder; PCR, CRS-PCR product (225 bp), Lane 1, carrier (225 bp and 201 bp); Lanes 2–4, non-carriers (201 bp). (D) Image of the PCR-PIRA amplicons on 3% agarose gel after digestion with PstI enzyme. L, 100 bp ladder; PCR, PCR-PIRA products (287 bp); Lanes 1 and 3, carriers (287 bp and 264 bp); Lanes 2 and 4, non-carriers (264 bp). Non-carrier sample 2 was misidentified as a complex vertebral malformation (CVM) carrier by AS-PCR. CVM-carrier sample 3 was misidentified as a non-carrier by CRS-PCR. Among the three methods, only the results of PCR-PIRA method were consistent with the results of DNA sequencing. w, wild-type allele; m, mutant allele.

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