9. Polymerase chain reaction (10 points) 5\" ATGTATTATTATTTTTTTGTTTTTTTTGCAATATA
ID: 276667 • Letter: 9
Question
9. Polymerase chain reaction (10 points) 5" ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATA AAGATCCTAACATTTTTGCGAG TAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAAC AAATGGAAGTTTATATTTAAATA GTGATTTTAATTTAAATGAAGCATCCAACGAAAGCTTCTTAGAAAATTGCAATATCA ATAGTTGTGTAGATATAGGTCAT GAAAATGGCAACAAAATAAATAGTCAAGAAAATGAGCATGCTAAAAATAATAACA ACAGTAATAATAACAATTTAAAACC AGAATACAATAATAATAATAATAATTTAAAACCAGAATACAATAATAATAATTTAA AACCAGAGTACAATAATAACAATT-3" One strand of a chromosomal DNA sequence is shown above. How would you amplify and isolate a DNA fragment defined by the sequence shown in red, using polymerase chain reaction. Design two PCR primers (Forward and Reverse primers, each 20 nucleotides long, that can be used to amplify the DNA segment. a) b) You have two restriction enzymes; HindIII and EcoRI that will be used for RFLP analysis of the amplified DNA. Which of the two enzymes can be used to digest the DNA? Show the restriction site on the DNA and show where the enzyme cleaves.Explanation / Answer
a) Forward primer is designed for the minus strand or the 3'-5' strand whereas reverse primer is designed for the plus strand or the 5'-3' strand. Both primers have 5’-3’ direction. The given sequence is in 5’-3’, so first we make its complimentary sequence which will be in the anti-parallel direction or 3’-5’ direction.
Forward primer: 5’ATGTATTATTATTTTTTTGT’3 , this will bind to the 3’-5’ strand
Reverse primer: 3’TCTCATGTTATTATTGTTAA’5, this primer will bind to the 5’-3’ strand
Given strand:
5’ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATAAAGATCCTAACATTTTTGCGAGTAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAACAAATGGAAGTTTATATTTAAATAGTGATTTTAATTTAAATGAAGCATCCAACGAAAGCTTCTTAGAAAATTGCAATATCAATAGTTGTGTAGATATAGGTCATGAAAATGGCAACAAAATAAATAGTCAAGAAAATGAGCATGCTAAAAATAATAACAACAGTAATAATAACAATTTAAAACCAGAATACAATAATAATAATAATAATTTAAAACCAGAATACAATAATAATAATTTAAAACCAG3’TC
AGTACAATAATAACAATT-3’
TCATGTTATTATTGTTAA’5
(Reverse primer)
Complimentary strand:
3’TACATAATAATAAAAAAACAAAAAAAACGTTA….’5
5’ATGTATTATTATTTTTTTGT’3
Forward primer
b) the given sequence has a hind III recognition sequence – 5’A|AGCTT’3
3’TTCGA|A’5
The vertical lines represent the sites where the enzyme cleaves the sequnece; the sequence is underlined and bolded in the following sequence
5’ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATAAAGATCCTAACATTTTTGCGAGTAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAACAAATGGAAGTTTATATTTAAATAGTGATTTTAATTTAAATGAAGCATCCAACGAA|AGCTTCTTAGAAAATTGCAATATCAATAGTTGTGTAGATATAGGTCATGAAAATGGCAACAAAATAAATAGTCAAGAAAATGAGCATGCTAAAAATAATAACAACAGTAATAATAACAATTTAAAACCAGAATACAATAATAATAATAATAATTTAAAACCAGAATACAATAATAATAATTTAAAACCAGAGTACAATAATAACAATT-3’
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