Question

1) Imagine that you are interested in studying the enzyme dihydrofolate reductase (DHFR) from Mycobacterium tuberculosis (this enzyme is a potential drug target). The amino acid sequence of the protein is: MTMVGCIWAQATSGVIGRGGDIPWRLPEDQAHFREITMGHTIVMGRRTWDSLPAKVRPLPGRRNWL SRQADFMASGAEWGSLEEALTSPETWVIGGGQVYALALPYATRCEVTEVDIGLPREAGDALAPVLD ETWRGETGEWRFSRSGLRYRLYSYHRS The DNA sequence within the M. tuberculosis genome that codes for the protein is: ATGACGATGGTGGGGCTGATCTGGGCTCAAGCGACATCGGGTGTCATCGGCCGCGGCGGCGACATCCCCT GGCGCTTGCCCGAGGACCAGGCGCATTTCCGGGAGATCACCATGGGGCACACGATCGTGATGGGCCGGCG CACATGGGATTCGCTGCCGGCTAAAGTCCGGCCGCTGCCCGGCCGGCGAAATGTCGTACTGAGCCGCCAA GCTGACTTTATGGCCAGCGGGGCTGAGGTTGTCGGTTCACTCGAGGAGGCGCTGACCAGCCCGGAGACGT GGGTGATCGGAGGCGGACAAGTCTATGCGCTGGCGCTGCCGTACGCGACCAGATGTGAGGTTACCGAGGT CGACATCGGCCTGCCGCGCGAAGCCGGTGACGCGCTGGCCCCCGTGCTGGACGAGACATGGCGGGGCGAG ACGGGGGAGTGGCGCTTCAGCCGGTCCGGGTTGCGGTACCGGTTGTACAGCTACCACCGCTCATGA Assume that you have been given a sample of the bacterial genomic DNA, restriction enzymes Ndel and BamHi, and a plasmid from Novagen called PET-28b. The PET-28b plasmid is commonly used to express proteins in E. coli. Information about the pET-28b plasmid appears in a 2-page PDF file on the class web page. You may also find additional regarding PET-28b on the web. (a) (2 pts) What nucleotide sequences are recognized by the restriction enzymes Ndel and BamHI? 1 311

Answer 1

a. The recognizing sequence is the restriction site  where the restriction enzyme recognize the site and cleave the restriction fragments. the sequence may vary based upon the enzyme.

Ndel sequence is: 5'CATATG3'

3'GTATAC5'

BamH1 sequence is: 5'GGATCC

3'CCTAGG

b.) PCR is a polymerize chain reaction where it needs a pair of primers to amplify the given template and resulting in the formation of product.

Forward primer: GCCTCCGGTAAGTCCGGCCGCT

Reverse primer :CGCGGCAGCGCGCGAGTGGCCGA

melting temperature can be estimated by formula: Tm = 4(G+C)/2(A+T)