Question

If you were asked to design PCR primers to amplify this gene, provide in the space below the sequences (5’ à 3’) of both the forward and reverse (sense and antisense) primers required to amplify the gene. Design your primers to be exactly 15 bases in length each. Design them so that the forward primer extreme 5’ end matches up with the extreme end of the start codon, and the reverse primer extreme 5’ end matches up with the extreme end of the stop codon.

Forward 5’ à 3’ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___

Reverse   5’ à 3’ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___

2572201 aaaaatagtt tacgatcgta aaaatctgca tatcatgata agagtggtta cattgccacg

2572261 ctgcttaacc cgccgatgcg cgggtttttt tgtacccaga atcctgtgag ctatacggaa

2572321 agtacacaga aaggaaggtg cgaccacaat taataacaaa atcttaaaaa ttgcacatgg

2572381 cactattagt tttctaaata ttgtgtattt tttgtattgc aggatgaccc tgtaacgaag

2572441 tttgcgtaac agcattttgc tctacgagtt tgccagcctc ccccagtggc tggctttttt

2572501 atgtccgtaa catcctgtgt atcaataaat gttgttgtct acgtacgtca agtagtcgca

2572561 tgagatctga ccagatatgt taaggttgca gctctctttg aatataatta tcattttcat

2572621 tacgttattg ttacgtttat ccggtgcgcc gtaaaacgcc gtccttcagg gggtggagga

2572681 tgtcaagaat atagttatcg tatggtgctc aaggagtatt gtgtaatatg aaaataatta

2572741 tttttagagt gctaactttt ttctttgtta tcttttcagt taatgtggtt gcgaaggaat

2572801 ttaccttaga cttctcgact gcaaagacgt atgtagattc gctgaatgtc attcgctctg

2572861 caataggtac tccattacag actatttcat caggaggtac gtctttactg atgattgata

2572921 gtggcacagg ggataatttg tttgcagttg atgtcagagg gatagatcca gaggaagggc

2572981 ggtttaataa tctacggctt attgttgaac gaaataattt atatgtgaca ggatttgtta

2573041 acaggacaaa taatgttttt tatcgctttg ctgatttttc acatgttacc tttccaggta

2573101 caacagcggt tacattgtct ggtgacagta gctataccac gttacagcgt gttgcaggga

2573161 tcagtcgtac ggggatgcag ataaatcgcc attcgttgac tacttcttat ctggatttaa

2573221 tgtcgcatag tggaacctca ctgacgcagt ctgtggcaag agcgatgtta cggtttgtta

2573281 ctgtgacagc tgaagcttta cgttttcggc aaatacagag gggatttcgt acaacactgg

2573341 atgatctcag tgggcgttct tatgtaatga ctgctgaaga tgttgatctt acattgaact

2573401 ggggaaggtt gagtagtgtc ctgcctgatt atcatggaca agactctgtt cgtgtaggaa

2573461 gaatttcttt tggaagcatt aatgcaattc tgggaagcgt ggcattaata ctgaattgtc

2573521 atcatcatgc atcgcgagtt gccagaatgg catctgatga gtttccttct atgtgtccgg

2573581 cagatggaag agtccgtggg attacgcaca ataaaatatt gtgggattca tccactctgg

2573641 gggcaattct gatgcgcaga actattagca gttgaggggg taaaatgaaa aaaacattat

2573701 taatagctgc atcgctttca tttttttcag caagtgcgct ggcgacgcct gattgtgtaa

2573761 ctggaaaggt ggagtataca aaatataatg atgacgatac ctttacagtt aaagtgggtg

2573821 ataaagaatt atttaccaac agatggaatc ttcagtctct tcttctcagt gcgcaaatta

2573881 cggggatgac tgtaaccatt aaaactaatg cctgtcataa tggaggggga ttcagcgaag

2573941 ttatttttcg ttgacttaga atagctcagt gaaaatagca ggcggagatt cataaatgtt

2574001

Answer 1

Forward Primer: 5’ aatctgcatatcatg 3’

Reverse primer: 5’ ttatgaatctccgcc 3’

Since start codon is AUG, so while designing the forward primer we need to focus on first finding the very first ATG in the sequence. We can design primers including this region in primer.

Similarly while designing reverse primer for the sequence we need to find the lastest of either TAA, TGA, or TAG which are corresponding sequences of UAA, UGA or UAG respectively as stop codons.

These sequences have been highlighted in the picture below.