Homework Sourse5 TCAGATAAAAAACTGGCACGCAATCTGCAATTAGCAAGACATCTTTTTAGAACACGCT
5’ -TCAGATAAAAAACTGGCACGCAATCTGCAATTAGCAAGACATCTTTTTAGAACACGCTGAATAAATTGAGGTTGCTATGTCTATTGTGGTGAAAAATAACATTCATTGGGTTGGTCAACGTGACTGGGAAGTGCGTGATTTTCACGGCACGGAATATAAAACGCTGCGCGGCAGCAGCTACAATAGCTACCTCATCCGCGAAGAAAAAAACGTGCTGATCGACACCGTCGACCATAAATTCAGCCGCGAATTTGTGCAGAACCTGCGTAATGAAATCGATCTGGCGGATATCGATTACATCGTGATTAACCATGCAGAAGAGGACCACGCTGGGGCGCTGACCGAACTGATGGCACAAATTCCCGATACGCCGATCTACTGTACAGCCAACGCTATCGACTCGATAAATGGTCATCACCATCATCCGGAGTGGAATTTTAATGTGGTGAAAACTGGCGACACGCTGGATATCGGCAACGGCAAACAGCTCATTTTTGTCGAAACACCAATGCTGCACTGGCCGGACAGCATGATGACTTACCTGACAGGCGACGCGGTGCTGTTCAGTAACGATGCTTTCGGTCAACACTACTGCGACGAGCATCTGTTCAACGATGAAGTGGATCAGACGGAGCTTTTCGAGCAGTGCCAGCGTTACTACGCCAATATCCTGACGCCGTTCAGCCGCCTGGTAACACCGAAAATTACCGAGATCCTGGGCTTTAACTTACCAGTCGATATGATAGCCACTTCCCACGGCGTGGTATGGCGCGATAACCCGACGCAAATTGTCGAGCTGTACCTGAAATGGGCGGCTGATTATCAGGAAGACAGAATCACCATTTTCTACGACACCATGTCGAATAACACCCGCATGATGGCTGACGCTATCGCCCAGGGGATTGCGGAAACCGACCCACGCGTGGCGGTGAAAATTTTCAACGTCGCCCGAAGCGATAAAAACGAAATCCTGACTAATGTCTTCCGCTCAAAAGGCGTGCTGGTCGGCACTTCGACGATGAATAACGTGATGATGCCGAAAATCGCCGGGCTGGTGGAGGAGATGACTGGTTTACGCTTCCGTAACAAACGCGCCAGTGCTTTCGGCTCTCACGGCTGGAGCGGCGGTGCGGTGGATCGTCTTTCCACGCGCCTGCAGGATGCGGGTTTCGAAATGTCGCTTAGCCTGAAAGCGAAATGGCGACCAGACCAGGACGCTCTGAAGTTATGCCGTGAACACGGTCGCGAAATCGCCCGTCAGTGGGCGCTCGCGCCGCTGCCGCAGAGCACGGTGAATACGGTAGTTAAAGAAGAAACCTCTGCCACCACGACGGCTGACCTCGGCCCACGGATGCAGTGCAGCGTCTGCCAGTGGATTTACGATCCGGCAAAAGGCGAGCCAATGCAGGACGTTGCGCCAGGAACGCCGTGGAGTGAAGTCCCGGATAACTTCCTCTGCCCGGAATGCTCCCTCGGCAAAGACGTCTTTGAAGAACTGGCATCGGAGGCAAAATGAGTAACGGCATTGTGATCATCGGTTCGGGCTTCGCCGCCCGCCAACTGGTGAAAAATATTCGCAAACAGGACGCCACTATTCCATT-3’
You wish to perform Quikchange mutagenesis on the gene of interest If you correctly translated the DNA sequence, the 194th amino acid encoded should be a tyrosine when you include the start methionine in your count. Design a primer that would allow you to convert tyrosine 194 to phenylalanine Y194F substitution). In addition to changing the amino acid identity, your primer should include at least 12-15 nucleotides on either side of the introduced changes, would ideally start and end with either a C or G, be between 25 and 45 nucleotides long and have a Tm of at least 78 oC. (Tm 81.5 0.41(%GC) (675 mismatch: and N primer length in nucleotides) a) What is the sequence of the forward primer you propose for this experiment? b) What is the Tm of the primer you designed? List the website or method you used for calculating the Tm since the calculated value depends on the method c) What is the sequence of the reverse primer you would employ to perform this Quikchange experiment? Remember to write your primers in the 5 3\' direction)Solution
A) Forward primer:
5\'-cgagcagtgccagcgtttctacgccaatatcctg-3\'
B) Tm calculation from the online tool is given below:
From NEB Tm calculation tool:
Nucleotides: 34
56% GC
Tm: 78 °C
TM calculator of applied biosciences
Tm of the Primer- 78.67
C) Sequence of the reverse primer:
5\'- caggatattggcgtagaaacgctggcactgctc-3\'
Workbook of the above ans:
Provided DNA sequence which can code for mRNA:
atg tct att gtg gtg aaa aat aac att cat tgg gtt ggt caa cgt gac tgg gaa gtg cgt gat ttt cac ggc acg gaa tat aaa acg ctg cgc ggc agc agc tac aat agc tac ctc atc cgc gaa gaa aaa aac gtg ctg atc gac acc gtc gac cat aaa ttc agc cgc gaa ttt gtg cag aac ctg cgt aat gaa atc gat ctg gcg gat atc gat tac atc gtg att aac cat gca gaa gag gac cac gct ggg gcg ctg acc gaa ctg atg gca caa att ccc gat acg ccg atc tac tgt aca gcc aac gct atc gac tcg ata aat ggt cat cac cat cat ccg gag tgg aat ttt aat gtg gtg aaa act ggc gac acg ctg gat atc ggc aac ggc aaa cag ctc att ttt gtc gaa aca cca atg ctg cac tgg ccg gac agc atg atg act tac ctg aca ggc gac gcg gtg ctg ttc agt aac gat gct ttc ggt caa cac tac tgc gac gag cat ctg ttc aac gat gaa gtg gat cag acg gag ctt ttc gag cag tgc cag cgt tac tac gcc aat atc ctg acg ccg ttc agc cgc ctg gta aca ccg aaa att acc gag atc ctg ggc ttt aac tta cca gtc gat atg ata gcc act tcc cac ggc gtg gta tgg cgc gat aac ccg acg caa att gtc gag ctg tac ctg aaa tgg gcg gct gat tat cag gaa gac aga atc acc att ttc tac gac acc atg tcg aat aac acc cgc atg atg gct gac gct atc gcc cag ggg att gcg gaa acc gac cca cgc gtg gcg gtg aaa att ttc aac gtc gcc cga agc gat aaa aac gaa atc ctg act aat gtc ttc cgc tca aaa ggc gtg ctg gtc ggc act tcg acg atg aat aac gtg atg atg ccg aaa atc gcc ggg ctg gtg gag gag atg act ggt tta cgc ttc cgt aac aaa cgc gcc agt gct ttc ggc tct cac ggc tgg agc ggc ggt gcg gtg gat cgt ctt tcc acg cgc ctg cag gat gcg ggt ttc gaa atg tcg ctt agc ctg aaa gcg aaa tgg cga cca gac cag gac gct ctg aag tta tgc cgt gaa cac ggt cgc gaa atc gcc cgt cag tgg gcg ctc gcg ccg ctg ccg cag agc acg gtg aat acg gta gtt aaa gaa gaa acc tct gcc acc acg acg gct gac ctc ggc cca cgg atg cag tgc agc gtc tgc cag tgg att tac gat ccg gca aaa ggc gag cca atg cag gac gtt gcg cca gga acg ccg tgg agt gaa gtc ccg gat aac ttc ctc tgc ccg gaa tgc tcc ctc ggc aaa gac gtc ttt gaa gaa ctg gca tcg gag gca aaa
Translated amino acid sequence: The underlined bold amino acid was changed (Y194F)
1- M S I V V K N N I H W V G Q R D W E V R D F H G T E Y K T L R G S S Y N S Y L I R E E K N V L I D T V D H K F S R E F V Q N L R N E I D L A D I D Y I V I N H A E E D H A G A L T E L M A Q I P D T P I Y C T A N A I D S I N G H H H H P E W N F N V V K T G D T L D I G N G K Q L I F V E T P M L H W P D S M M T Y L T G D A V L F S N D A F G Q H Y C D E H L F N D E V D Q T E L F E Q C Q R Y Y A N I L T P F S R L V T P K I T E I L G F N L P V D M I A T S H G V V W R D N P T Q I V E L Y L K W A A D Y Q E D R I T I F Y D T M S N N T R M M A D A I A Q G I A E T D P R V A V K I F N V A R S D K N E I L T N V F R S K G V L V G T S T M N N V M M P K I A G L V E E M T G L R F R N K R A S A F G S H G W S G G A V D R L S T R L Q D A G F E M S L S L K A K W R P D Q D A L K L C R E H G R E I A R Q W A L A P L P Q S T V N T V V K E E T S A T T T A D L G P R M Q C S V C Q W I Y D P A K G E P M Q D V A P G T P W S E V P D N F L C P E C S L G K D V F E E L A S E A K-479
1- M S I V V K N N I H W V G Q R D W E V R D F H G T E Y K T L R G S S Y N S Y L I R E E K N V L I D T V D H K F S R E F V Q N L R N E I D L A D I D Y I V I N H A E E D H A G A L T E L M A Q I P D T P I Y C T A N A I D S I N G H H H H P E W N F N V V K T G D T L D I G N G K Q L I F V E T P M L H W P D S M M T Y L T G D A V L F S N D A F G Q H Y C D E H L F N D E V D Q T E L F E Q C Q R F Y A N I L T P F S R L V T P K I T E I L G F N L P V D M I A T S H G V V W R D N P T Q I V E L Y L K W A A D Y Q E D R I T I F Y D T M S N N T R M M A D A I A Q G I A E T D P R V A V K I F N V A R S D K N E I L T N V F R S K G V L V G T S T M N N V M M P K I A G L V E E M T G L R F R N K R A S A F G S H G W S G G A V D R L S T R L Q D A G F E M S L S L K A K W R P D Q D A L K L C R E H G R E I A R Q W A L A P L P Q S T V N T V V K E E T S A T T T A D L G P R M Q C S V C Q W I Y D P A K G E P M Q D V A P G T P W S E V P D N F L C P E C S L G K D V F E E L A S E A K-479
Designed primer from the above sequence (in bold and underlined):
cgagcagtgccagcgttactacgccaatatcctg
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3\'-gctcgtcacggtcgcaaagatgcggttataggac-5\'
5\'-cgagcagtgccagcgtttctacgccaatatcctg-3\'
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gctcgtcacggtcgcaatgatgcggttataggac
Forward primer: 5\'- cgagcagtgccagcgtttctacgccaatatcctg- 3\'
Reverse primer: 3\'-gctcgtcacggtcgcaaagatgcggttataggac-5\'
5\'- caggatattggcgtagaaacgctggcactgctc -3\'
