Page 43 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

Solved Problems 337

b. If you prepared genomic DNA from a tissue sam- molecules in which the pBS281 DNA has been joined
ple containing millions of cells, would the frag- with a fragment of human DNA. Answer the follow-
ments produced by partial digestion of DNA from ing questions concerning the junction between the
all of these cells be the same or different? two different kinds of DNA.
10. The text stated that molecular biologists have devel- a. What proportion of the junctions between pBS281
oped elegant techniques that can convert any type of and all possible human DNA fragments can be
DNA end into any other type of DNA end. In this cleaved with MboI?
problem, consider genomic DNA that is broken by b. What proportion of the junctions between pBS281
mechanical shearing into random pieces. Some of the and all possible human DNA fragments can be
ends of these pieces are blunt, some have 5′-over- cleaved with BamHI?
hangs, and others have 3′-overhangs. c. What proportion of the junctions between pBS281
a. Must the two ends of any one genomic DNA and all possible human DNA fragments can be
fragment be of the same type? cleaved with XorII (5′ C^GATCG 3′)?
b. Explain why the ends with 5′ or 3′ overhangs are d. What proportion of the junctions between pBS281
not sticky. and all possible human DNA fragments can be
c. Researchers can convert ends with overhangs into cleaved with BstYI (5′ R^GATCY 3′)? (R and Y
blunt ends using either DNA polymerase (plus the stand for purine and pyrimidine, respectively.)
four dNTPs), or nuclease S1, which degrades e. What proportion of all possible junctions that can
single-stranded regions of DNA but not double- be cleaved with BamHI will result from cases in
stranded regions. Which kinds of ends with over- which the cleavage site in human DNA was not a
hangs (5′ or 3′) could be converted into blunt ends BamHI site in the human chromosome?
using DNA polymerase? With S1 nuclease?
15. A recombinant DNA molecule is constructed using a
plasmid vector called pMBG36 that is 4271 bp long.
Section 9.2 The pMBG36 plasmid contains a so-called polylinker
11. a. What is the purpose of molecular cloning? that has a single site for each of several restriction
b. What purpose do selectable markers serve in vectors? enzymes, including BamHI (5′ G^GATCC 3′) and
EcoRI (5′ G^AATTC 3′). The sequence of the poly-
c. What is the purpose of the origin of replication in a linker region of pMBG36 is shown below; the dots in-
plasmid vector? dicate the large majority of the vector that is not
d. Why do cloning vectors have polylinkers? shown. You now cut pMBG36 with EcoRI and
12. Which of the enzymes from the following list would insert into it a fragment of the DNA previously
you need to make a recombinant DNA molecule? What shown in Problem 4 that is also cut with EcoRI.
is the function of those enzyme(s) in the process?
5'. . .C G G AT CC CC TA AGA TG A AT TC CGCGCGC AT CG GC. . . 3'
a. DNA polymerase 3' . . .G C C TA GG GG AT TCT AC T TA AG GCGCGCG TA GC C G. . .5'
b. RNA polymerase a. Write out as much of the DNA sequence of the re-
c. A restriction enzyme sultant recombinant DNA molecule as is possible.
d. DNA ligase Two answers are possible; you need to show
e. An aminoacyl-tRNA synthetase only one.
f. Peptidyl transferase b. Why are there two possible answers to part (a)?
g. Reverse transcriptase c. How many recognition sites for BamHI will be
13. Is it possible that two different restriction enzymes could found in the recombinant DNA molecule shown
in your answer to part (a)?
cut the human genome into exactly the same number of
fragments and with exactly the same distribution of frag- d. If you cut this recombinant DNA molecule with
ment sizes, yet the ends produced by the two enzymes BamHI and run the digest on a gel, how many
could not be joined together by DNA ligase? Explain. bands would you see and how large would they be?
14. A plasmid vector pBS281 is cleaved by the enzyme e. How many recognition sites for EcoRI will be
BamHI (5′ G^GATCC 3′), which recognizes only one found in the recombinant DNA molecule shown in
site in the DNA molecule. Human DNA is digested your answer to part (a)?
with the enzyme MboI (5′ ^GATC 3′), which recog- f. If you cut this recombinant DNA molecule with
nizes many sites in human DNA. These two digested EcoRI and run the digest on a gel, how many bands
DNAs are now ligated together. Consider only those would you see and how large would they be?