Below is some sequence around the region of the PER3 gene that are amplified by

Question

Below is some sequence around the region of the PER3 gene that are amplified by PCR, which contains five of the 54bp repeats.

TCCTATCACA GGCTGGAAGC AATAAGACAG CTCAAAATTT TATGACACTA CCAGAATGGC

TGACAATTTT AAACTTATGA ACTGTTTATT TCTGGAATTT TCCATTTACT ATTTTTGGAC

CTTGGTTGAC CACAGGTAAC TGAAACTACA GAAAGCAAAA CCATGAATAA AGGAGGACTA

CTGTATTTTG TGATAAGAAG ATTAAAGTGT CTTTTCATGT GCCCTTACTT TCTAGCAGTG

TGTTACAGGC AACAATGGCA GTGAGAGCAG TCCTGCTACT ACCGGTGCAC TGTCCACGGG

GTCACCTCCC AGGGAGAATC CATCCCATCC TACTGCCAGC GCTCTGTCCA CAGGATCGCC

TCCCATGAAG AATCCATCCC ATCCTACTGC CAGCGCTCTG TCCACAGGAT CGCCTCCCAT

GAAGAATCCA TCCCATCCTA CTGCCAGCAC ACTGTCCATG GGATTGCCTC CCAGCAGGAC

TCCATCCCAT CCTACTGCCA CTGTTCTGTC CACGGGGTCA CCTCCCAGCG AATCCCCATC

CAGAACTGGT TCAGCAGCAT CAGGTAGTGG ATCAGGACAA CTAATGTTTC AAACTCCAAT

GCCAGACATT CACTATGTGC TGAGCTCTCA CTGTGTGCCC CAGGCACTGA TGTGGAAGTA

CAAGGTTTTT TTTTCTTTTT CCCTTTTTCC TTTTTGTCAG GTATATTGGG GTATATTTAT

ACACAATAAA ATTCACCAGT TTGAGGGGTA CAAACAAGAA TTCTGACAGA TGTGTACCTT

AGTGTAAGCG CTACCACAAT CACCATA

The 2 primers used in this PCR were as follows:

PER3F 5' CAAAATTTTATGACACTACCAGAATGGCTGAC 3'

PER3R 5' AACCTTGTACTTCCACATCAGTGCCTGG 3'

What would the size of the PCR product be if the individual who supplied the DNA template was homozygous for the five repeat allele?

Explanation / Answer

First check the sequence where the 5' Forward primer match....so in our 1 and 2 sequences

1. TCCTATCACA GGCTGGAAGC AATAAGACAG CT F- 5' CAAAATTT TATGACACTA CCAGAATGGC

2. TGAC 3' AATTTT AAACTTATGA ACTGTTTATT TCTGGAATTT TCCATTTACT ATTTTTGGAC

So the bolded and the underlined sequence matches with the forward 5' end so our sequence starts from the middle of the seq1 to first few nucleotides of the seq2.

Now do the complementary of 5'---3' sequence of the reverse primer.

R -5' AACCTTGTACTTCCACATCAGTGCCTGG 3'

3' TTGGAACATGAAGGTGTAGTCACGGACC 5'

Now search for the 3' TTGGAACATGAAGGTGTAGTCACGGACC 5' Sequence by reversing the sequence that is from 5' CCAGGC...................GGTT 3' In our sequences.

We can find this exact sequence in

GCCAGACATT CACTATGTGC TGAGCTCTCA CTGTGTGCC R- 5'C CAGGCACTGA TGTGGAAGTA

CAAGGTT 3' TTT TTTTCTTTTT CCCTTTTTCC TTTTTGTCAG GTATATTGGG GTATATTTAT

Count the number of nucleotides present inbetween the two primer sequences including the primer sequence that will be the size of the PCR product.

SSRs found in your sequence(s)

Sequence Motif No.of Repeats SSR start SSR end SeqLength

TCCTATCACA-1 gt 3 188 193 757

TCCTATCACA-2 tg 3 582 587 757

Sequence - The identifying tag given to the sequence in title line. The appendage ("-1," "-2," etc.) is an index number which identifies the first, second, etc. SSR found in a particular sequence.
Motif - the simple sequence that is repeated.
No. of Repeats - number of times the simple sequence (motif) is repeated.
SSR start - start coordinate of the SSR. The number is nth nucleotide from the beginning of the sequence.
SSR end - end coordinate of the SSR. The number is nth nucleotide from the beginning of the sequence + the length of the SSR.
SeqLength - the total length of the sequence in which the SSR was found.

Now accordingly we have to design a primer for the above two sequences.

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