Question

You now plan to amplify a 770 bp region of this gene, highlighted in grey below, which includes the full coding sequence plus some flanking regions. Design a primer set (forward & reverse primers of 20 nucleotide each) that will allow you to amplify only the sequence highlighted in grey in your PCR reaction. (2)

AGCCAACTCAAGGATTTAACAGCGACAACAACGCATTACACCTTTTCTGGCAGGACGGAGATAAAGTCTT ACCGCTTGAGCGCAAAGAGCTCCTTGGCCAATTATTACTCGACGAGATCGTGACCCGTTATGATGAAAAA AATCGACGTTAAGATTCTGGACCCGCGCGTTGGGAAGGAATTTCCGCTCCCGACTTATGCCACCTCTGGC TCTGCCGGACTTGACCTGCGTGCCTGTCTCAACGACGCCGTAGAACTGGCTCCGGGTGACACTACGCTGG TTCCGACCGGGCTGGCGATTCATATTGCCGATCCTTCACTGGCGGCAATGATGCTGCCGCGCTCCGGATT GGGACATAAGCACGGTATCGTGCTTGGTAACCTGGTAGGATTGATCGATTCTGACTATCAGGGCCAGTTG ATGATTTCCGTGTGGAACCGTGGTCAGGACAGCTTCACCATTCAACCTGGCGAACGCATCGCCCAGATGA TTTTTGTTCCGGTAGTACAGGCTGAATTTAATCTGGTGGAAGATTTCGACGCCACCGACCGCGGTGAAGG CGGCTTTGGTCACTCTGGTCGTCAGTAACACATACGCATCCGAATAACGTCATAACATAGCCGCAAACAT TTCGTTTGCGGTCATAGCGTGGGTGCCGCCTGGCAAGTGCTTATTTTCAGGGGTATTTTGTAACATGGCA GAAAAACAAACTGCGAAAAGGAACCGTCGCGAGGAAATACTTCAGTCTCTGGCGCTGATGCTGGAATCCA GCGATGGAAGCCAACGTATCACGACGGCAAAACTGGCCGCCTCTGTCGGCGTTTCCGAAGCGGCACTGTA TCGCCACTTCCCCAGTAAGACCCGCATGTTCGATAGCCTGATTGAGTTTATCGAAGATAGCCTGATTACT

(note: primers should not extend beyond the grey region)

Fp:

Rp:

NB: DNA is double stranded and the sequence above is that of the coding (sense) strand only. Your primers need to be designed so that they are complementary to their respective binding sites, and the Fp should bind to the one strand while the Rp binds to the other strand. a) What is the Tm of your forward and reverse primers respectively? (2) Fp: Rp:

b) Based on the last two criteria for primer design outlined in question (4) above, do your primers meet the criteria? Explain. (3)

c) What would be the consequence of setting the PCR machine to an annealing temperature lower than the melting temperatures (Tm) of the primer set?

Answer 1

	Sequence (5'->3')	Template strand	Length	Start	Stop	Tm	GC%	Self complementarity	Self 3' complementarity
Forward primer	ACCGCTTGAGCGCAAAGAGC	Plus	20	1	20	64.26	60.00	6.00	4.00
Reverse primer	TACAGTGCCGCTTCGGAAAC	Minus	20	770	751	60.95	55.00	5.00	5.00
Product length	770

Fp: Ans: ACCGCTTGAGCGCAAAGAGC

Rp: Ans: TACAGTGCCGCTTCGGAAAC

a)

Ans: Tm of Fp: 64.26ο C and Tm of Rp: 60.95ο C

b) Based on the last two criteria for primer design outlined in question (4) above, do your primers meet the criteria? Explain.

Ans: the criteria for primer design outlined in question (4) are not provided here in present question, So comparison is not possible until and unless the criteria is provided. However, in table I have provided all the necessary information required to decide the primer efficiency. So that you can compare this information with primer outline in question (4). In general Tm difference in two primers (Fp and Rp) should not have more than 5ο C difference. GC % should be between 50-60% etc.   

c) What would be the consequence of setting the PCR machine to an annealing temperature lower than the melting temperatures (Tm) of the primer set?

Ans: if we set the PCR PCR machine setting to an annealing temperature lower than the Tm, The set of primers will annealed (bind to the DNA) to the gene of interest with complimentary base pairing. (General rule is Ta, Annealing Temperature should be less than Tm, Melting Temperature for better annealing of primers)