Question

The sequence of the cDNA of the human beta-globin chain is shown below:

ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAA CGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCC AGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCT AAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCT GGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACG TGGATCCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCAC TTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGT GGCTAATGCCCTGGCCCACAAGTATCACTAA

The start codon is shown in red and the stop codon is shown in blue.

An undergraduate student wants to clone this cDNA into the pGEX-2T vector in frame with the GST-Thrombin tag:

2

Analysis of the cDNA sequence in a DNA analysis software, revealed the restriction sites of the most common restriction enzymes:

1) Which restriction site do you need to use to insert the full beta-globin cDNA sequence from start to stop codon?

Ans:
2) In one sentence, explain why:

Ans:

3) Design 28-30 nt long primers to amplify the cDNA by PCR for cloning into pGEX- 2T in frame with the GST-Thrombin tag:

P1: P2:

Answer 1

1. Any two restriction enzymes which are not present in gene and vector; and present in MCS of vector can be chosen.

Eg. XmaI and PfoI (as it is not present in the gene)

2. Because it is not present in gene and vector (other than MCS); and present in MCS of vector. In other word, these are unique cutters.

3.

Random seq RE site gene sequence

P1: ACATATCCCGGGATGGTGCATCTGACT

P2: ACATATTCCNGGATTAGTGATACTTGTG