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Paper Information

Journal:   GENETICS IN THE 3RD MILLENNIUM   SPRING 2016 , Volume 14 , Number 2; Page(s) 4256 To 4261.
 
Paper: 

OPTIMAL CONDITIONS FOR AMPLIFICATION OF MICROSATELLITE LENGTH POLYMORPHISMS SUCH AS POLY (A) IN 3' UTR OF VDR GENE

 
 
Author(s):  HOSSIENZADEH COLAGAR ABASALT*, MORADI FIROUZJAH HAMID, KARIMIAN MOHAMMAD
 
* DEPARTMENT OF MOLECULAR AND CELL BIOLOGY, FACULTY OF BASIC SCIENCES, UNIVERSITY OF MAZANDARAN, BABOLSAR, IRAN
 
Abstract: 

Optimization of the condition for PCR-directed sequencing of microsatellites poly Adenine (A) length polymorphisms is more difficult and sensitive compared with other common sequences. Replication slippage may occur for polymerase enzyme during microsatellite amplification and direct sequencing of these PCR products will be challenging for heterozygote samples. So, the aim of this study is to introduce optimal condition for amplification of microsatellites poly (A) length polymorphisms such as variation of poly (A) at the 3?-end of the vitamin D receptor (VDR) gene. In this studyfirstly, we analyzed various types of microsatellite sequences by in silico method to select a short microsatellite sequence which has various alleles with a little length difference.
Finally, VDR poly (A) microsatellite was selected. Amplification of this locus was optimized by applying hot-start PCR using gradients of temperature, DMSO, MgCl2, Taq polymerase, and primers concentration.
Crush and soak method was used for DNA extraction of alleles in heterozygote samples from polyacrylamide gel. PCR products were analyzed by using single-strand conformation polymorphism and PCR-direct sequencing.
Our observations showed that hot-start PCR was optimized in the concentration of 0.2 mM of primers, 0.8mM of MgCl2 and 1.75 unit of Taq polymerase in 25?l PCR mixtures. These optimal conditions lead to remove Taq polymerase slippage and non-specific PCR product. Considering the possible errors in amplification and analysis of microsatellite, the presented method in this study could be an efficient strategy in analysis of these sequences.

 
Keyword(s): MICROSATELLITE REPEATS, POLYMERASE CHAIN REACTION, SHORT SEQUENCE REPEATS, POLYMORPHISM, SINGLE-STRANDED CONFORMATIONAL
 
 
References: 
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Citations: 
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+ Click to Cite.
APA: Copy

HOSSIENZADEH COLAGAR, A., & MORADI FIROUZJAH, H., & KARIMIAN, M. (2016). OPTIMAL CONDITIONS FOR AMPLIFICATION OF MICROSATELLITE LENGTH POLYMORPHISMS SUCH AS POLY (A) IN 3' UTR OF VDR GENE. GENETICS IN THE 3RD MILLENNIUM, 14(2), 4256-4261. https://www.sid.ir/en/journal/ViewPaper.aspx?id=508681



Vancouver: Copy

HOSSIENZADEH COLAGAR ABASALT, MORADI FIROUZJAH HAMID, KARIMIAN MOHAMMAD. OPTIMAL CONDITIONS FOR AMPLIFICATION OF MICROSATELLITE LENGTH POLYMORPHISMS SUCH AS POLY (A) IN 3' UTR OF VDR GENE. GENETICS IN THE 3RD MILLENNIUM. 2016 [cited 2021August02];14(2):4256-4261. Available from: https://www.sid.ir/en/journal/ViewPaper.aspx?id=508681



IEEE: Copy

HOSSIENZADEH COLAGAR, A., MORADI FIROUZJAH, H., KARIMIAN, M., 2016. OPTIMAL CONDITIONS FOR AMPLIFICATION OF MICROSATELLITE LENGTH POLYMORPHISMS SUCH AS POLY (A) IN 3' UTR OF VDR GENE. GENETICS IN THE 3RD MILLENNIUM, [online] 14(2), pp.4256-4261. Available: https://www.sid.ir/en/journal/ViewPaper.aspx?id=508681.



 
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