Question

1. Below is the sequence for the SARS-CoV-2 spike protein that encodes a 1757 amino acid protein capable of binding surface receptors on some cell types. The nucleotide sequence is from base pair 21,563 to 25,384 in the viral genome (numbers in left-most column). Upon binding, the virus is uptaken into the cell, the coat is shed, and viral RNA released into the cytoplasm. When this occurs, host cell ribosomes begin transcription and translation of the RNA, including this protein. In order to study its binding capabilities to host cell receptors, you want to clone the gene into a plasmid designed for protein expression and purification. Design forward and reverse primers that will successfully bind the targeted gene sequence and amplify the gene. The primers should be 12-15 nucleotides long. The sequence has been reverse transcribed (RNA to DNA) and presented in the 5’ to 3’ format below. Bold or underline the nucleotides you will use to design your primers. Label the 5’ and 3’ ends of the primers.

21563-atgtttgt ttttcttgtt ttattgccac tagtctctag

    21601 tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac

    21661 acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga

    21721 cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggac

    21781 caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc

    21841 ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa

    21901 gacccagtcc ctacttattg ttaataacgc tactaatgtt gttattaaag tctgtgaatt

    21961 tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat

    22021 ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca

    22081 gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt

    22141 gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt

    22201 gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat

    22261 taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga

    22321 ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag

    22381 gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact

    22441 tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta

    22501 tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac

    22561 aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg

    22621 gaacaggaag agaatcagca actgtgttgc tgattattct gtcctatata attccgcatc

    22681 attttccact tttaagtgtt atggagtgtc tcctactaaa ttaaatgatc tctgctttac

    22741 taatgtctat gcagattcat ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg

    22801 gcaaactgga aagattgctg attataatta taaattacca gatgatttta caggctgcgt

    22861 tatagcttgg aattctaaca atcttgattc taaggttggt ggtaattata attacctgta

    22921 tagattgttt aggaagtcta atctcaaacc ttttgagaga gatatttcaa ctgaaatcta

    22981 tcaggccggt agcacacctt gtaatggtgt tgaaggtttt aattgttact ttcctttaca

    23041 atcatatggt ttccaaccca ctaatggtgt tggttaccaa ccatacagag tagtagtact

    23101 ttcttttgaa cttctacatg caccagcaac tgtttgtgga cctaaaaagt ctactaattt

    23161 ggttaaaaac aaatgtgtca atttcaactt caatggttta acaggcacag gtgttcttac

    23221 tgagtctaac aaaaagtttc tgcctttcca acaatttggc agagacattg ctgacactac

    23281 tgatgctgtc cgtgatccac agacacttga gattcttgac attacaccat gttcttttgg

    23341 tggtgtcagt gttataacac caggaacaaa tacttctaac caggttgctg ttctttatca

    23401 ggatgttaac tgcacagaag tccctgttgc tattcatgca gatcaactta ctcctacttg

    23461 gcgtgtttat tctacaggtt ctaatgtttt tcaaacacgt gcaggctgtt taataggggc

    23521 tgaacatgtc aacaactcat atgagtgtga catacccatt ggtgcaggta tatgcgctag

    23581 ttatcagact cagactaatt ctcctcggcg ggcacgtagt gtagctagtc aatccatcat

    23641 tgcctacact atgtcacttg gtgcagaaaa ttcagttgct tactctaata actctattgc

    23701 catacccaca aattttacta ttagtgttac cacagaaatt ctaccagtgt ctatgaccaa

    23761 gacatcagta gattgtacaa tgtacatttg tggtgattca actgaatgca gcaatctttt

    23821 gttgcaatat ggcagttttt gtacacaatt aaaccgtgct ttaactggaa tagctgttga

    23881 acaagacaaa aacacccaag aagtttttgc acaagtcaaa caaatttaca aaacaccacc

    23941 aattaaagat tttggtggtt ttaatttttc acaaatatta ccagatccat caaaaccaag

    24001 caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt

    24061 catcaaacaa tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca

    24121 aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata

    24181 cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgc

    24241 attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca

    24301 gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa

    24361 aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa

    24421 ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat

    24481 ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat

    24541 tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc aacaattaat

    24601 tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt

    24661 acttggacaa tcaaaaagag ttgatttttg tggaaagggc tatcatctta tgtccttccc

    24721 tcagtcagca cctcatggtg tagtcttctt gcatgtgact tatgtccctg cacaagaaaa

    24781 gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg

    24841 tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca

    24901 aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt

    24961 caacaacaca gtttatgatc ctttgcaacc tgaattagac tcattcaagg aggagttaga

    25021 taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa

    25081 tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt

    25141 aaatgaatct ctcatcgatc tccaagaact tggaaagtat gagcagtata taaaatggcc

    25201 atggtacatt tggctaggtt ttatagctgg cttgattgcc atagtaatgg tgacaattat

    25261 gctttgctgt atgaccagtt gctgtagttg tctcaagggc tgttgttctt gtggatcctg

    25321 ctgcaaattt gatgaagacg actctgagcc agtgctcaaa ggagtcaaat tacattacac

    25381 ataa

Answer 1

The sequences used for the designing of forward and reverse primer are underlined below.

5' 21563-atgtttgt ttttcttgtt ttattgccac tagtctctag

    21601 tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac

    21661 acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga

    21721 cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggac

    21781 caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc

    21841 ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa

    21901 gacccagtcc ctacttattg ttaataacgc tactaatgtt gttattaaag tctgtgaatt

    21961 tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat

    22021 ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca

    22081 gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt

    22141 gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt

    22201 gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat

    22261 taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga

    22321 ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag

    22381 gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact

    22441 tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta

    22501 tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac

    22561 aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg

    22621 gaacaggaag agaatcagca actgtgttgc tgattattct gtcctatata attccgcatc

    22681 attttccact tttaagtgtt atggagtgtc tcctactaaa ttaaatgatc tctgctttac

    22741 taatgtctat gcagattcat ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg

    22801 gcaaactgga aagattgctg attataatta taaattacca gatgatttta caggctgcgt

    22861 tatagcttgg aattctaaca atcttgattc taaggttggt ggtaattata attacctgta

    22921 tagattgttt aggaagtcta atctcaaacc ttttgagaga gatatttcaa ctgaaatcta

    22981 tcaggccggt agcacacctt gtaatggtgt tgaaggtttt aattgttact ttcctttaca

    23041 atcatatggt ttccaaccca ctaatggtgt tggttaccaa ccatacagag tagtagtact

    23101 ttcttttgaa cttctacatg caccagcaac tgtttgtgga cctaaaaagt ctactaattt

    23161 ggttaaaaac aaatgtgtca atttcaactt caatggttta acaggcacag gtgttcttac

    23221 tgagtctaac aaaaagtttc tgcctttcca acaatttggc agagacattg ctgacactac

    23281 tgatgctgtc cgtgatccac agacacttga gattcttgac attacaccat gttcttttgg

    23341 tggtgtcagt gttataacac caggaacaaa tacttctaac caggttgctg ttctttatca

    23401 ggatgttaac tgcacagaag tccctgttgc tattcatgca gatcaactta ctcctacttg

    23461 gcgtgtttat tctacaggtt ctaatgtttt tcaaacacgt gcaggctgtt taataggggc

    23521 tgaacatgtc aacaactcat atgagtgtga catacccatt ggtgcaggta tatgcgctag

    23581 ttatcagact cagactaatt ctcctcggcg ggcacgtagt gtagctagtc aatccatcat

    23641 tgcctacact atgtcacttg gtgcagaaaa ttcagttgct tactctaata actctattgc

    23701 catacccaca aattttacta ttagtgttac cacagaaatt ctaccagtgt ctatgaccaa

    23761 gacatcagta gattgtacaa tgtacatttg tggtgattca actgaatgca gcaatctttt

    23821 gttgcaatat ggcagttttt gtacacaatt aaaccgtgct ttaactggaa tagctgttga

    23881 acaagacaaa aacacccaag aagtttttgc acaagtcaaa caaatttaca aaacaccacc

    23941 aattaaagat tttggtggtt ttaatttttc acaaatatta ccagatccat caaaaccaag

    24001 caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt

    24061 catcaaacaa tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca

    24121 aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata

    24181 cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgc

    24241 attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca

    24301 gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa

    24361 aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa

    24421 ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat

    24481 ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat

    24541 tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc aacaattaat

    24601 tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt

    24661 acttggacaa tcaaaaagag ttgatttttg tggaaagggc tatcatctta tgtccttccc

    24721 tcagtcagca cctcatggtg tagtcttctt gcatgtgact tatgtccctg cacaagaaaa

    24781 gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg

    24841 tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca

    24901 aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt

    24961 caacaacaca gtttatgatc ctttgcaacc tgaattagac tcattcaagg aggagttaga

    25021 taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa

    25081 tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt

    25141 aaatgaatct ctcatcgatc tccaagaact tggaaagtat gagcagtata taaaatggcc

    25201 atggtacatt tggctaggtt ttatagctgg cttgattgcc atagtaatgg tgacaattat

    25261 gctttgctgt atgaccagtt gctgtagttg tctcaagggc tgttgttctt gtggatcctg

    25321 ctgcaaattt gatgaagacg actctgagcc agtgctcaaa ggagtcaaat tacattacac

    25381 ataa 3'

The forward primer will be exactly same as the sequences at the 5' end of the DNA. Because during DNA amplification the double stranded DNA will be separated and the forward primer binds to the 3' end of the complementary strand of the given DNA strand as the synthesis of DNA will always be in a direction from 5' to 3'. Always the bonds between G and C is better that A and G, because G and C binds with three hydrogen bonds and the other have only two hydrogen bonds between them. The 13th nucleotide on the 5' end of the given DNA is 'C', according to the instructions the primer length should be between 12-15. So, the the primer with better binding will have 13 nucleotides.

The forward primer will be : 5' atgtttgtttttc 3'

The reverse primer will bind to the 3' end of the given DNA sequence. Therefore, the primer will be complemetary to the DNA sequences at the 3 end. Here, the complementary sequence will be 3' AATGTAATGTGTATT 5'. The reverse primer is the reverse complement, hence, the reverse primer is 5' TTATGTGTAATGTAA 3'.