3\'CCTGAAGTATGGATTACGCGTATGAGCCGATAATGAAATGAT5\'. For determining the sequence o

Question

3'CCTGAAGTATGGATTACGCGTATGAGCCGATAATGAAATGAT5'. For determining the sequence of the DNA molecule shown above by the chain termination method: a) Give the sequence of a 12 base primer b) What components, in addition to the primer, will be needed for the "G" c) Show the sequences of all the DNA products in the "G" reaction tube. d) What DNA lengths will be produced in the "G" reaction?. e) What DNA lengths will be produced in the "T" reaction? f Your plan is to clone the fragment into a cloning vector with a PstI site, recognition sequence CTCA1G. How could you design PCR primers to accomplish this goal? Give the sequences.*

Explanation / Answer

pstPrimer sequence is

5’ GGACTTCATACC 3’

In G reaction apart from primer, all the dNTPs and ddGTP is required. Apart from that DNA template, buffer, enzyme is also required.

Reaction will start after primer and for it will stop at G . In chain termination reaction amount of ddGTP is adjusted in such a way so that it chances of ddGTP and dGTP incorporation is also possible. So that it will be readable.

5’GGACTTCATACCTAATG 3’

5’GGACTTCATACCTAATGCG 3’

5’GGACTTCATACCTAATGCGCATACTCG 3’

5’GGACTTCATACCTAATGCGCATACTCGG 3’

For T-reaction

GGACTTCATACCT

GGACTTCATACCTAAT

GGACTTCATACCTAATGCGCAT

GGACTTCATACCTAATGCGCATACT

GGACTTCATACCTAATGCGCATACTCGGCT

GGACTTCATACCTAATGCGCATACTCGGCTAT

GGACTTCATACCTAATGCGCATACTCGGCTATT

GGACTTCATACCTAATGCGCATACTCGGCTATTACT

GGACTTCATACCTAATGCGCATACTCGGCTATTACTT

GGACTTCATACCTAATGCGCATACTCGGCTATTACTTT

GGACTTCATACCTAATGCGCATACTCGGCTATTACTTTACT

In this question you have to remember that complimentary DNA has been synthesized, hence all the sequence is complimentary.

Pst I is staggered end cutter and second two primers are required to clone the DNA, here as per question I am showing only one primer.

Here sequence of complimentary strand is

5’GGACTTCATACCTAATGCGCATACTCGGCTATTACTTTACTA3’

That starts with G, here use the template G and add restion site.

Restriction sequence is

5’CTGCAG3’

3’GACGTC 5’

That sequence will incorporate in compliment DNA so that the direction of DNA will be same.

In this primer some bases will be added to before to restriction site so that enzyme will get the appropriate binding.

5’CCAATTCTGCAGGACTTCATACC3’

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