How can we demonstrate that the gene we have isolate is indeed the one for prote

Question

How can we demonstrate that the gene we have isolate is indeed the one for protein P?

1. The ampicillin-resistance gene for plasmid pBR322 contains a single PstI restriction site. Genes that are inserted into an amp-r region are high expressed. If an antibody specific for protein P is available, how would we now construct our gene library and detect the clones of interest?

3. After we have cloned the gene for protein P into the ampr region of pBR322, suppose that we had to rely only on the drug-resistance properties of the plasmid to select from the gene library those cells carrying the plasmid plus the gene for protein P. What drug-resistance characteristic would the desired cells exhibit?

4. how would you select for tet-r, amp-s cell?

5. suppose that the base sequence of the gene for protein P is known. A defective protein P has been discovered, and its gene has also been cloned and sequenced. Both genes are 1000 bp in length and are flanked on either side by a site (-GAATTC-) for the restriction enzyme EcoRI. A base sequence GCATTC- exists 300 bp from one end of the normal gene for protein P, but the sequence -GAATTC- exists at that position in the abnormal gene. We want to find out if fetal cells contain the normal gene. So we cleave DNA from fetal cells with EcoRI and separate the fragments on an agarose gel. A probe for the normal gene will also hybridize with abnormal gene if it differes only a few bases from the normal genes. The size of the fetal DNA fragments can be estimated by running DNA fragments of known sizes on the same gel. What band pattern is expected if the fetal cells contain the abnormal gene?

Explanation / Answer

Ans.1- pBR322 plasmid contains ampR amd tetR gene. three single cutter restriction sites are there PstI, EcoRI and BhamH1. we can insert our gene of interest between PstI and EcoRI. We can use Tetracycline for selection.

Ans 3- Tetracycline can be used for selection

Ans 4- Tetracycline selection

Ans- After digestion of abnormal cells with EcoRI we will get two bands -300bp and -700bp but in case of normal cell we will will get only single 1kb size band

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