Effect of single nucleotide mismatch at 3′-end of the primers in selected candidate micro RNA breast cancer genes

Paper Details

Research Paper 01/07/2016
Views (203) Download (7)
current_issue_feature_image
publication_file

Effect of single nucleotide mismatch at 3′-end of the primers in selected candidate micro RNA breast cancer genes

Aftab Ali Shah, Mushtaq Ahmed, Farman Ullah
Int. J. Biosci.9( 1), 430-434, July 2016.
Certificate: IJB 2016 [Generate Certificate]

Abstract

Polymerase chain reaction (PCR) is an important biological tool for qualitative and quantitative detection and amplification of desired sequence of DNA (deoxyribonucleic acid) in living organisms. The success of PCR reaction is based on the specificity of the primers used. The potential of these primers can be influenced by primer-template DNA, primer-primer complementarily, annealing, extension temperatures and nature of the mismatch. But, often the powers of thermal cycler are restricted due to primer–template mismatches which can lead to inaccuracies/false results. In the present study, the effect of single nucleotide mismatches (No change, T/A and C/G) at the 3′-end of primers on PCR reaction of breast cancer candidate microRNA genes i.e; hsa-mir-196a-2 (1), hsa-mir-196a-2 (2) and hsa-mir-146a respectively was evaluated in the human genome using conventional PCR. Here we conclude that no effect was found on the PCR product of these studied genes with introduced single mismatches at the 3′-end of primers under standard PCR conditions.

VIEWS 2

Bru D, Martin-Laurent F, Philippot L. 2008. Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appled Enviro-nmental Microbiology 74, 1660-1663. http:// dx.doi. org/10.1128/aem.02403-07.

Christopherson C, Sninsky JJ, Kwok S. 1997. The effects of internal primer template mismatches on RT-PCR: hIV-1 model studies. Nucleic Acids Research 25, 654-658. http://dx.doi.org/10.1093/ nar/25.3.654.

Garcia AI, Buisson M, Bertrand P, Rimokh R, Rouleau E, Lopez BS, Lidereau R, Mikaélian I, Mazoyer S. 2011. Down‐regulation of BRCA1 expression by mi R‐146a and mi R‐146b‐5p in triple negative sporadic breast cancers, EMBO molecular medicine 3, 279-290. http://dx.doi.org/10.1002/ emmm. 201100136.

Huang M, Arnheim N, Goodman MF. 1992. Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Research 20, 4567-4573. http://dx.doi.org/10.1093/nar/20.17.4567.

Hu Z, Liang J, Wang Z, Tian T, Zhou X, Chen J, Miao R, Wang Y, Wang X, Shen H. 2009. Common genetic variants in pre‐micro RNAs were associated with increased risk of breast cancer in Chinese women, Human mutation 30, 79-84.  http://dx.doi.org/10.1002/humu.20837.

Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Ménard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM. 2005. Micro RNA gene expression deregulation in human breast cancer, Cancer research 65, 7065-7070. http://dx.doi.org/10.1158/0008-5472.can-05-1783.

Jedlinskia DJ, Gabrovskaa PN, Weinsteina SR, Smitha RA, Griffithsa LR. 2011. Single nucleotide polymorphism in hsa-mir-196a-2 and breast cancer risk: a case control study, Twin Research and Human Genetics 14, 417-421. http://dx.doi.org/10.1375/twin.14.5.417.

Kalle, E., Kubista, M. and Rensing, C. 2014. Multi-template polymerase chain reaction, Biomo-lecular Detection and Quantification  2, 11-29. http:/ /dx. doi.org/10.1016/j.bdq.2014.11.002.

Kung JT, Colognori D, Lee JT. 2013. Long noncoding RNAs: past, present, and future, Genetics 193, 651-669. http://dx.doi.org/10.1534/genetics. 112. 146704.

Kwok S, Kellogg DE, McKinney N, Spasic D, Godal L, Levenson C, Sninsky JJ. 1990. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Research 18, 999-1005. http://dx.doi.org/10.1093/nar/18.4.999.

Qiu MT, Hu JW, Ding XX, Yang X, Zhang Z, Yin R, Xu L. 2012. Hsa-mi R-499 rs 3746444 polymorphism contributes to cancer risk: a meta-analysis of 12 studies, PLoS One 7, e50887. http://dx.doi.org/10.1371/journal.pone.0050887.

Rickham PP. 1964. Human Experimentation. Code of Ethics of the World Medical Association. Declaration of Helsinki, British medical journal 2, 177. http://dx.doi.org/10.1136/bmj.2.5402.177.

Simsek M, Adnan H. 2000. Effect of single mismatches at 3′–end of primers on polymerase chain reaction, Journal for scientific research. Medical sciences/Sultan Qaboos University 2, 11. http://dx.doi.org/10.18295/squmj/. 

Smith S, Vigilant L, Morin PA. 2002. The effects of sequence length and oligonucleotide mismatches on 5 exonuclease assay efficiency. Nucleic Acids Research 20, 43-49. http://dx.doi.org/10.1093/nar/gnf110.

Stadhouders R, Pas SD, Anber J, Voermans J, Mes THM, Schutten M. 2010. The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay. Journal of Molecular Diagnosis 12, 109-117. http://dx.doi.org/10.2353/jmoldx.2010.090035. 

Thalia A. Farazi, Jessica I. Hoell, Pavel Morozov, Thomas Tuschl. 2013. MicroRNAs in human cancer. In, MicroRNA Cancer Regulation. Springer pp. 1-20. http://dx.doi.org/10.1007/978-94-007-5590-11.

Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM. 2006 A micro RNA expression signature of human solid tumors defines cancer gene targets, Proceedings of the National academy of Sciences of the United States of America 103, 2257-2261. http://dx.doi.org/10.1073/pnas.0510565103.