Page 40 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

334 Chapter 9 Digital Analysis of DNA

WHAT’S NEXT

The Human Genome Project did not end with the determina- functions of these proteins and RNAs? What kinds of DNA
tion of 3 billion base pairs of DNA sequence. An essential part sequences make up other important features of chromosomes
of the project’s task was to make sense of this vast amount of such as centromeres and telomeres? In the next chapter, we
information. Where in all of these As, Cs, Gs, and Ts are the explain how scientists identified functional elements of the
genes? What do the sequences of genes predict about the genome and how their findings revealed, at the level of DNA
kinds of proteins and RNAs the genes encode and the possible sequence, the architecture of the human genome.

SOLVED PROBLEMS

I. The following map of the plasmid cloning vector only if the insert fragment joins with the cut vector
pBR322 shows the locations of the ampicillin (amp) DNA. The cut vector will not be able to re-ligate
and tetracycline (tet) resistance genes as well as two without an inserted fragment because the BamHI and
unique restriction enzyme recognition sites, one for EcoRI sticky ends are not complementary and cannot
EcoRI and one for BamHI. You digested this plasmid base pair. All ampicillin-resistant colonies therefore
vector with both EcoRI and BamHI enzymes and pu- contain a BamHI-EcoRI fragment of human DNA
rified the large EcoRI–BamHI vector fragment. You ligated to the BamHI-EcoRI sites of the vector. Frag-
also digested human genomic DNA that you want to ments cloned at the BamHI-EcoRI sites interrupt and
insert into the vector with both EcoRI and BamHI. therefore inactivate the tetracycline resistance gene.
After mixing the plasmid vector and the human ge- All ampicillin-resistant clones will be tetracycline
nomic fragments together and ligating, you trans- sensitive.
formed an ampicillin-sensitive strain of E. coli and b. If the gene for ampicillin resistance contained an
selected for ampicillin-resistant colonies. EcoRI site while the tetracycline resistance gene had
EcoRI a BamHI site, the cloning process would destroy the
BamHI
activity of both genes. Thus, you would be unable to
amp r select bacterial cells transformed with recombinant
tet r DNA molecules.
c. The primers would have to flank the human genomic
DNA inserts (pink) as shown in the following diagram.
a. If you test all of your selected ampicillin-resistant EcoRI
transformants for tetracycline resistance, what result Human DNA insert
do you expect, and why?
b. Why is it important that the EcoRI site not be located Primer 2
in the gene conferring ampicillin resistance?
BamHI
c. Diagram the positions and orientations of two oligo- r
nucleotide primers that you could use to sequence the amp Primer 1
two ends of the human DNA insert found in any re-
combinant DNA molecule made by this method.
d. Why would a library made in this fashion represent
less than one genome equivalent? d. By chance, some regions of the human genome
would have two EcoRI sites in a row without a
Answer BamHI site in between. Other regions would have
This problem requires an understanding of vectors and the two BamHI sites in a row without an EcoRI site in
process of combining DNAs using sticky ends generated the middle. When such regions are cut with both en-
by restriction enzymes. zymes, the resultant fragments would not have the
two different kinds of sticky ends required for inser-
a. The plasmid must be circular to replicate in E. coli, tion into the vector cut with both enzymes. Thus,
and in this case, a circular molecule will be formed many regions of the human genome could not be
DNA: © Design Pics/Bilderbuch RF cloned into the vector by this procedure.