Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA.

M. K. Rosli, A. S. Zamzuriada, S. M. Syed-Shabthar, M. C. Mahani, O. Abas-Mazni, Badrul Munir Md. Zain

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.

Original languageEnglish
Pages (from-to)2554-2568
Number of pages15
JournalGenetics and molecular research : GMR
Volume10
Issue number4
Publication statusPublished - 2011

Fingerprint

Cytochromes b
Electron Transport Complex IV
Mitochondrial DNA
rRNA Genes
Polymerase Chain Reaction
Taq Polymerase
Oxidoreductases
Temperature
ribosomal RNA 12S
DNA
Genes
Buffers

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA. / Rosli, M. K.; Zamzuriada, A. S.; Syed-Shabthar, S. M.; Mahani, M. C.; Abas-Mazni, O.; Md. Zain, Badrul Munir.

In: Genetics and molecular research : GMR, Vol. 10, No. 4, 2011, p. 2554-2568.

Research output: Contribution to journalArticle

Rosli, M. K. ; Zamzuriada, A. S. ; Syed-Shabthar, S. M. ; Mahani, M. C. ; Abas-Mazni, O. ; Md. Zain, Badrul Munir. / Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA. In: Genetics and molecular research : GMR. 2011 ; Vol. 10, No. 4. pp. 2554-2568.
@article{03544b8be856455698a8d0c7a6d126b0,
title = "Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA.",
abstract = "PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.",
author = "Rosli, {M. K.} and Zamzuriada, {A. S.} and Syed-Shabthar, {S. M.} and Mahani, {M. C.} and O. Abas-Mazni and {Md. Zain}, {Badrul Munir}",
year = "2011",
language = "English",
volume = "10",
pages = "2554--2568",
journal = "Genetics and Molecular Research",
issn = "1676-5680",
publisher = "Fundacao de Pesquisas Cientificas de Ribeirao Preto",
number = "4",

}

TY - JOUR

T1 - Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA.

AU - Rosli, M. K.

AU - Zamzuriada, A. S.

AU - Syed-Shabthar, S. M.

AU - Mahani, M. C.

AU - Abas-Mazni, O.

AU - Md. Zain, Badrul Munir

PY - 2011

Y1 - 2011

N2 - PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.

AB - PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.

UR - http://www.scopus.com/inward/record.url?scp=84856419948&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84856419948&partnerID=8YFLogxK

M3 - Article

VL - 10

SP - 2554

EP - 2568

JO - Genetics and Molecular Research

JF - Genetics and Molecular Research

SN - 1676-5680

IS - 4

ER -