The following DNA sequence from 5end to 3end is for the acti

The following DNA sequence, from 5\'end to 3\'end, is for the actin binding Rvsl67 protein. ATGAGTTTTA AGATACAATG GCCAGCAAGG TGTGTTTCCT GGGAACTACG TGCAACTCAA CAAGAACTAG 3\' Design forward and reverse primers, consisting of 10 bases, required for PCR amplification of Rvsl67 gene.

Solution

Hi,

5’ ATGAGTTTTA AGA-----------CAA CAAGAACTAG 3’

This is the sequence given to you. In order to PCR amplify the gene of interest, you need to design primers which will bind to the upper and lower ends of the given segment. The most crucial aspect of the problem is that the DNA is double stranded and only one strand is given to you. SO you will have to write it in dsDNA format. Then design one primer for upper strand and another for lower strand. The primers should run in opposite direction so as to amplify the gene.

1. Make it double stranded – write complimentary sequence.

5’ ATGAGTTTTA AGA-----------CAA CAAGAACTAG 3’

3’ TACTCAAAAT TCT ------------ GTT GTTCTTGATC 5’

2. Primers. The forward primer is complementary to the lower strand in 5’à 3’ direction. And the reverse primer is complementary to the upper strand in 5’ to 3’ direction (reverse complement)

                                                      3’ GTT GTTCTTGATC 5’

5’ ATGAGTTTTA AGA-----------CAA CAAGAACTAG 3’

3’ TACTCAAAAT TCT ------------ GTT GTTCTTGATC 5’

5’ ATGAGTTTTA 3\'

So two primers are: forward= 5’ ATGAGTTTTA 3’

                                       Reverse = 5’ CTAGTTCTTG 3’

in simple terms, forward primer = same as the start of sequence

reverse primer = reverse complement of the end of the sequence.