Close
Help
Need Help?





JOURNAL

Biochemistry Insights

88,009 Journal Article Views | Journal Analytics

Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

Submit a Paper



Publication Date: 06 Nov 2008

Journal: Biochemistry Insights

Citation: Biochemistry Insights 2008:1 35-39

Erin Curry1, Scott L. Pratt1, Dale E. Kelley1, Daniel R. Lapin2 and John R. Gibbons1,2

1Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634. 2OvaMax, Inc. Watertown, WI 53094.

Abstract

A reliable and sensitive method of genetic analysis is necessary to detect multiple specific nucleic acid sequences from samples containing limited template. The most widely utilized method of specific gene detection, polymerase chain reaction (PCR), imparts inconsistent results when assessing samples with restricted template, especially in a multiplex reaction when copies of target genes are unequal. This study aimed to compare two methods of PCR product analysis, fluorescent detection following agarose gel electrophoresis or dot blot hybridization with chemiluminescent evaluation, in the detection of a single copy gene (SRY) and a multicopy gene (β-actin). Bovine embryo sex determination was employed to exploit the limited DNA template available and the target genes of unequal copies. Primers were used either independently or together in a duplex reaction with purified bovine genomic DNA or DNA isolated from embryos. When used independently, SRY and β-actin products were detected on a gel at the equivalent of 4-cell or 1-cell of DNA, respectively; however, the duplex reaction produced visible SRY bands at the 256 cell DNA equivalent and β-actin products at the 64 cell DNA equivalent. Upon blotting and hybridization of the duplex PCR reaction, product was visible at the 1–4 cell DNA equivalent. Duplex PCR was also conducted on 186 bovine embryos and product was subjected to gel electrophoresis or dot-blot hybridization in duplicate. Using PCR alone, sex determination was not possible for 22.6% of the samples. Using PCR combined with dot blot hybridization, 100.0% of the samples exhibited either both the male specific and β-actin products or the β-actin signal alone, indicating that the reaction worked in all samples. This study demonstrated that PCR amplification followed by dot blot hybridization provided more conclusive results in the evaluation of samples with low DNA concentrations and target genes of unequal copies.


Downloads

PDF  (644.31 KB PDF FORMAT)

RIS citation   (ENDNOTE, REFERENCE MANAGER, PROCITE, REFWORKS)

BibTex citation   (BIBDESK, LATEX)


Sharing




What Your Colleagues Say About Libertas Academica
As a new contributor, my experience publishing our article in Genetics and Epigenetics was extremely positive.  The reviewing process was prompt and fair (four reviewers examined our review article!) and then, the editorial office was very helpful by regularly updating me on the progression of the publication process.  I strongly recommend the journal to my colleagues in the field and hope that the journal will grow up and gain the notoriety it deserves in our ...
Dr Sylviane Muller (Institut de Biologie Moléculaire et Cellulaire, CNRS, Strasbourg, France)
More Testimonials

Quick Links


New article and journal news notification services
Email Alerts RSS Feeds
Facebook Google+ Twitter
Pinterest Tumblr YouTube