MBB 141 Questions 21-23

21. A cDNA library is made with mRNA isolated from liver tissue. When a cloned cDNA from that library is digested with the enzymes EcoRI (E), HindIII (H), and BamHI (B), the restriction map shown above, part (a) is obtained. When this cDNA is used to screen a cDNA library made with mRNA from brain tissue, three identical cDNAs with the restriction map show in the following figure, part (b) are obtained. When either cDNA is used to synthesize a uniformly labeled 32-P-labeled probe and the probe is allowed to hybridize to a Southern blot prepared from genomic DNA digested singly with the enzymes EcoRI, HindIII, and BamHI, an autoradiograph shows the pattern of bands in the figure part (c). When either cDNA is used to synthesize a uniformly labeled 32-P-labeled probe and used to probe a northern blot prepared with poly(A) RNA isolated from live and brain tissues, the pattern of bands in part (d) of the figure is seen. Fully analyze these data and then answer the following questions:

a) Do these cDNAs derive from the same gene?
b) Why are different-sized bands seen on the northern blot?
c) Why do the cDNAs have different restriction maps?
d) Why are some of the bands seen on the whole-genome Southern blot different sizes than some of the restriction fragments in the cDNAs?

22. Draw the pattern of bands you would expect to see on a DNA sequencing gel if you annealed the primer 5’-CTAGG-3’ to the following single-stranded DNA fragment and carried out a dideoxy sequencing experiment. Assume the dNTP precursors were all labeled.

3’-GATCCAAGTCTACGTATAGGCC-5’

23. Katrina purified a clone from a plasmid library made using genomic DNA and sequenced a 500-bp long segment using the dideoxy sequencing method. Her clone (twin sister) Marina used PCR with Taq DNA polymerase to amplify the same 500 bp fragment from genomic DNA. Marina sequenced the fragment using the dideoxy sequencing method, and obtained the same sequence as Katrina did. She then cloned the fragment into a plasmid vector, and, following ligation and transformation into E. coli, sequenced several, independently isolated plasmids to verify that she cloned the correct sequence. Most of them have the same sequence as Katrina’s clone, but Marina finds that about1/3 of them have a sequence that differs in one or two base pairs. None of the clones that differ from Katrina’s clone are identical. Fearing she has done something wrong, Marina repeats her work, only to obtain the same results: about 1/3 of the fragments cloned from the PCR product have single base differences. Explain this discrepancy.