An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region.

Mousumi Sahu,Ranjan Sarmah,Madhumita Barooah,Mahendra Kumar Modi,Smita Sahoo,Budheswar Dehury,Jagajjit Sahu,Priyabrata Sen,Kishore Sarma

doi:10.6026/97320630008181

Abstract

Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab.

Highlights

Classical methods for degenerate primer design include software applications such as Gene Fisher [1], CODEHOP [2] or PrimaClade [3]
The technique is based on multiple local alignments, using the Multiple Expectation – Maximization for Motif Elicitation (MEME) algorithm [5], in order to search for conserved regions long enough to serve as primers
Two motifs were generated using MAST by considering and combining evidences of all p-values associated with motifs resulted by MEME run.Primer3 was employed to generate primers for every individual sequence, which were compared with the common motif portions of that sequence Table 2 suggested by the first MEME result

Summary

Introduction

Classical methods for degenerate primer design include software applications such as Gene Fisher [1], CODEHOP [2] or PrimaClade [3]. These methods usually rely on the identification of clear blocks of conserved regions in multiple global alignments. In many cases, primer design using multiple global alignments is unsuccessful [4], due to poor conservation among the sequences under study. A method is analyzed which suggests the common motif region could be used to design degenerate of primers for sequences with poor global similarity or without well-conserved blocks in multiple global alignments. The potential primer properties of these motifs will be verified further in wet lab

Methods

Discussion

Conclusion