Designing Allele SA Bioinformati Designing Allele

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for primer designing like primer3, primer3 plus, prim blast, batchprimer3 (non-commercial software) and t like fast-PCR, primer Express (commercial software).
Designing Allele Specific Primers: A Bioinformatics Approach Tejas C Bosamia1, Hiren N. Bhalani1, Sahil V. Patel2, Pratibha Singh3, Abhay Kumar2 1

Dept. of Plant Biotechnology & Biotechnology, Junagadh Agricultural University, Junagadh Junagad 362 001, Gujarat; 2 Crop Improvement Unit, Directorate of Groundnut Research, Ivnagar Road, Junagadh 362 001, Gujarat; 3Dept. of Biochemistry, Indian Institute of Science, Bangalore, 560 012, Karnataka Primer designing is one of the most critical criteria for successful amplification of desired PCR product of DNA. A poorly designed primer could result in little or no desired amplicon. There are number of softwares available forr primer designing like primer3, primer3 plus, primer blast, batchprimer3 (non-commercial mmercial software) and that like fast-PCR, PCR, primer Express (commercial software). All these softwares have different rent qualities and functionalities, functionalities but majority of the softwares have the same algorithm for calculating melting temperature (Tm) of primer. Primer3 Pri is the most popular non-commercial commercial primer design software because of its capabilities and free accessibility to the users (You et al., 2008). Several molecular marker systems are available but simple imple sequence repeats (SSR) and single nucleotide polymorphism (SNP) are the two most important markers of choice in molecular breeding due to its co-dominant co nature (ability to distinguish heterozygous state of gene or a particular locus).. Such markers are employed in developing genetic linkage age map, QTL mapping mappin and marker-assisted ssisted selection. SSRs are multi locus while SNPs are bi-allelic allelic and most abundantly present in genome (Kalia et al., 2011).. Various SNP genotyping platforms are available including Taqman®, Amplifluor®, genome resequencing, and SNP arrays. However, all these methods require significant initial investments for expensive probes, micro-chips, chips, or special instrumentation (Liu et al., 2012). On the other hand, the Allele Specific pecific Primer (ASP) amplification method is relatively flexible and costeffective in nature (Hirotsu et al., 2010).

igure 1, 1 three primers are required alleles). As indicated in figure for detecting one allele. The common flanking primer is set for internal control and one specific s primer for each allele. So, at the end, two separate separa PCR reactions of three primers are required for detecting each allele viz. two common flanking primers and one allele specific primer. As shown inn figure 1, G allele specific primer amplifies only G allele and is similar for A allele specific primer.

AS primer (ASP) designing

The addition of artificial mismatches at the third base from 3'end of the primers might be added to increase the reliability of discrimination between two alleles (Hayashi ( et al., 2004). The reliability of ASP after adding secondary mismatch was demonstrated in Arabidopsis thaliana and wheat (Wei et al., ., 2009). Rules for selection of a

The Allele specific primers are designed in such a way that their 3' terminal nucleotides correspond to an SNP, matching perfectly with one allele (the specific allele) and having a 3' mismatch smatch with other alleles (the non-specific non

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Figure 1. Primer design of allele allel specific primers (figure adapted from You et al., 2008)

Allele specific primer extension is depending on the difference in extension efficiency of DNA polymerase between primers with matched and mismatched 3' ends. DNA polymerase extends a primer only when the 3' end is perfectly complementary plementary to the DNA template. Allele specific primers preferentially trigger amplification ampli of the specific allele and the presence of the SNP can be detected as PCR amplification after electrophoresis. elec Genotyping is based on determination of the primer that produces the amplicon. However, each of the ASP is a dominant marker, pairs of AS primers for both alleles can be used as co-dominant markers.

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nucleotide for the secondary mismatch are summarized in (Table 1). A 'strong' mismatch (G/A or C/T) at the 3'-end of an allele-specific primer will likely need a 'weak' second mismatch (C/A, or G/T) and vice-versa for other mismatches. Table 1: Rule for adding mismatch at 3’ end and secondary mismatch at third base position of 3’ end during designing AS primers Code of a SNP

Alleles of SNP

Mismatch at the 3’ end

Mismatch strength at 3’ end

R

G/A

G/T, A/C

Weak

Y

T/C

G/T, A/C

Weak

S

G/C

G/G, C/C

Medium

W

A/T

A/A , T/T

Medium

K

G/T

G/A, T/C

Strong

M

A/C

G/A, T/C

Strong

Secondary mismatch

G/A, T/C, A/G, C/T G/A, T/C, A/G, C/T C/C, A/A, G/G, T/T C/C, A/A, G/G, T/T G/T, A/C, T/G, C/A G/T, A/C, T/G, C/A

The ASP methodology allows SNP genotyping without expensive probes or specialized equipment. This method is highly versatile due to its simplicity and flexibility. This method could be established easily in any molecular biology laboratory.

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Reference Hayashi K., Hashimoto N., Daigen M., and Ashikawa I. (2004). Development of PCR- based SNP markers for rice blast resistance genes at the Piz locus. Theor. Appl. Genet., 108:1212-1220. Hirotsu N., Murakami N., Kashiwagi T., Ujiie K., and Ishimaru K. (2010). Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species. Plant Methods, 6:12. Kalia R.K., Rai M.K., Kalia S., Singh R. and Dhawan A.K. (2011). Microsatellite markers: an overview of the recent progress in plants. Euphytica, 177:309–334. Liu J., Huang S., Sun M., Liu S., Liu Y., Wang W., Zhang X., Wang H. and Hua W. (2012). An improved allelespecific PCR primer design method for SNP marker analysis and its application. Plant Methods, 8:34. Sobrino B., Brion M., and Carracedo A. (2005). SNPs in forensic genetics: a review on SNP typing methodologies. Forensic Sci. Int., 154:181-194. Wei B., Jing R., Wang C., Chen J., Mao X., Chang X., and Jia J. (2009). Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs. Mol. Breeding, 23:13-22. You F.M., Huo N., Gu Y.Q., Luo M., Ma Y., Hane D., Lazo G.R., Dvorak J. and Anderson O.D. (2008). BatchPrimer3: A high throughput web application for PCR and sequencing primer design. BMC Bioinformatics, 9:253.

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