Indan Journal of Medical Research Indan Journal of Medical Research Indan Journal of Medical Research Indan Journal of Medical Research
  Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login  
  Home Print this page Email this page Small font sizeDefault font sizeIncrease font size Users Online: 1097       
ORIGINAL ARTICLE
Year : 2015  |  Volume : 142  |  Issue : 5  |  Page : 555-562

Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR


1 Department of Paediatrics, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India
2 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India

Correspondence Address:
B Vishnu Bhat
Department of Paediatrics, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry 605 006
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-5916.171282

Rights and Permissions

Background & objectives: Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics. Methods: Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing. Results: The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (T m ) values. Variations in Ct and T m values among the two alleles were observed in both rs3014866 (Ct: C allele - 24±1, T allele - 27±1; T m : C allele - 82.5±0.3, T allele - 86.3±0.2) and rs2149356 (Ct: C allele - 24±1, A allele - 26±1; T m : C allele - 79.4±0.2, A allele - 80.4±0.3). Based on the variations, homozygous and heterozygous alleles were detected. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR. Interpretation & conclusions: In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping.


[FULL TEXT] [PDF]*
Print this article     Email this article
 Next article
 Previous article
 Table of Contents

 Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
 Citation Manager
 Access Statistics
 Reader Comments
 Email Alert *
 Add to My List *
 * Requires registration (Free)
 

 Article Access Statistics
    Viewed1295    
    Printed9    
    Emailed0    
    PDF Downloaded535    
    Comments [Add]    
    Cited by others 3    

Recommend this journal