Download Gene Isolation and Manipulation

Gene Isolation and Manipulation
4.
Ligase is an essential enzyme within all cells that seals breaks in the phosphatesugar backbone of DNA. During DNA replication it joins Okazaki fragments to
create a continuous strand, and in cloning, it is used to join the various DNA
fragments with the vector. If it was not added, the vector and cloned DNA would
simply fall apart.
6.
Each cycle takes five minutes and doubles the DNA. In one hour there would be
12 cycles so the DNA would be amplified 212 = 4096-fold amplification.
8.
plasmid (15–20 kb) < cosmid (35–45 kb) < BAC (150–300 kb) < YAC (300 kb
and larger)
15.
The commercial cloning of insulin was into bacteria. Bacteria are not capable of
processing introns. Genomic DNA would include the introns, while cDNA is a
copy of processed (and thus intron-free) mRNA.
16.
This problem assumes a random and equal distribution of nucleotides.
AluI (1/4 )4 = on average, once in every 256 nucleotide pairs
EcoRI (1/4 )6 = on average, once in every 4096 nucleotide pairs
AcyI (1/4)4( 1/2 )2 = on average, once in every 1024 nucleotide pairs
25.
Conservatively, the amount of DNA necessary to encode this protein of 445 amino
acids is 445 × 3 = 1335 base pairs. When compared with the actual amount of
DNA used, 60 kb, the gene appears to be roughly 45 times larger than necessary.
This “extra” DNA mostly represents the introns that must be correctly spliced
out of the primary transcript during RNA processing for correct translation.
(There are also comparatively very small amounts of both 5´ and 3´ untranslated
regions of the final mRNA that are necessary for correct translation encoded by
this 60-kb of DNA.)
32.
The region of DNA that encodes tyrosinase in “normal” mouse genomic DNA
contains two EcoRI sites. Thus, after EcoRI digestion, three different-sized
fragments hybridize to the cDNA clone. When genomic DNA from certain albino
mice is subjected to similar analysis, there are no DNA fragments that contain
complementary sequences to the same cDNA. This indicates that these mice lack
the ability to produce tyrosinase because the DNA that encodes the enzyme must
be deleted.