Lecture 31: RFLPs, DNA fingerprinting, polymerase chain reaction
1. What is meant by the term "restriction map"? What
is the value of using partial digests in such mapping?
2. What is the advantage of preparing a restriction map with more
than one restriction endonuclease?
3. Define restriction fragment length polymorphism (RFLP) and
describe the procedures that are used to detect RFLPs.
4. RFLP's are usually described in terms of a specific probe and
a specific restriction endonuclease. Would you expect to be able
to detect RFLPs with the same probe but different restriction
endonucleases? Explain your answer.
5. What type of probe would you use to detect an RFLP that is not located
within the coding sequence of a gene? What are such probes called and why
are they considered to be so valuable in studies seeking to identify
human disease genes?
of RFLPs?
6. What is an RFLP haplotype? What special value does it have
in human genetic analysis?
7. How do RFLP haplotypes contribute to the identification of
the genes responsible for specific human genetic diseases?
8. Describe three distinctly different types of genetic changes
that could alter the pattern of restriction fragment lengths observed
with Bam HI (G^GATCC) and a 5 kb probe. (^ = cut site)
9. How can the length of a restriction fragment be altered by
a polymorphic cut site that lies outside of the region that hybridizes
to the probe?
10. Probe X detects an RFLP that involves two polymorphic HindIII
sites.
b. How many different haplotypes would you expect to see in any
one individual and why?
c. How would you interpret an individual that exhibited only one
haplotype?
d. Would you expect Hin dIII to cut probe X into smaller pieces?
Explain the reasoning behind your answer.
e. What results would you expect in part a if you were using a probe based
on the first 50 nucleotides at the 5'-end of probe X? Explain your answer.
11. Probe Z is 4.0 kb in length. Use diagrams to explain how each
of the following RFLP patterns might be obtained with probe Z and
appropriate restriction endonucleases with six nucleotide recognition
sites.
b. a single fragment 4.2 kb in length vs. a fragment 7.3 kb in
length.
c. A haplotype consisting of fragments 4.7 and 5.1 kb in length
vs a haplotype consisting of fragments 2.5 and 4.7 kb in length.
d. A haplotype consisting of fragments 2.5, 4.7 and 5.1 kb in
length vs. a haplotype consisting of fragments 3.6 and 5.1 kb
in length. (Hint: think in terms of sampling the general population and
do not assume that the difference necessarily arises from a single
mutational event.)
e. What general pattern of fragments would you expect to see if
you used the same probe with a restriction endonuclease that has
a four nucleotide recognition site.
12. Using the table of codons in the textbook, identify all possible
amino acid sequences whose genomic coding sequences could generate
a cut site for EcoRI (G|AATTC). (Be sure to examine all possible
reading frames and exclude all nucleotide sequences that could
not be found in the coding sequences for proteins.)
13. In an organism whose DNA is 50% AT, how long would a protein
have to be to have a 50% chance of containing a cut site for EcoRI
within its coding sequence?
14. Describe the genetic defect that is responsible for Huntington
disease.
15. Discuss the psychological problems that must be considered
before deciding whether or not the child of a known Huntington
disease parent should be screened to determine whether he or she
has inherited the disease. Also answer for a young adult whose
father has just begun to develop the symptoms of Huntington disease.
16. What feature makes genetic screening for Huntington disease
unusually easy?
17. How can Huntington disease patients with two normal parents
be explained? What types of tests would you perform if the husband
of the mother of the patient demanded proof that he was actually
the father of the patient?
18. The gene that becomes altered in Huntington disease and the
nature of the changes that lead to the disease have been identified
precisely. Describe the additional problems that stand in the
way of finding a cure for the disease.
19. "Annonymous" probes are now widely used in human
genetic studies.
b. What is the advantage of using annonymous probes rather than
known genes?
c. Describe the role played by an annonymous probe in the identification
of the gene that is altered in Huntington disease.
d. What special feature was possessed by the G8 annonymous probe
used in the study of Huntington disease that made it potentially
more valuable than a simpler annonymous probe might have been?
e. Would you expect the same G8 haplotype to be associated with
Huntington disease in all disease-prone families? Explain your
answer.
20. Explain how "exon-trapping" is used to identify
genes within an uncharacterized segment of chromosomal DNA.
21. Briefly summarize techniques that can be used to identify
active genes within a chromosomal segment that has been shown
by linkage analysis to contain the gene responsible for a human
inherited disease.
22. How would you determine which of the active genes identified
in question 21 was the one responsible for the genetic disease
under study?
23. What characteristic of the restriction endonuclease Hpa
II causes it to yield "tiny" fragments (as well as unusually
large fragments) when compared to most of the commonly used restriction
endonucleases? How were these properties exploited in the search
for the cystic fibrosis gene?
24. Explain how detailed genetic analysis of female patients with
Duchenne muscular dystrophy (DMD) made it possible to identify
the chromosomal region that carried the gene that is defective
in DMD.
25. Mutations in gene BRCA1 are responsible for much of human
hereditary breast cancer. What practical problems stand in the
way of developing an effective genetic screening program for carriers
of the defective genes?
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a. How many different haplotypes would be seen in a survey of
a population that includes polymorphisms at both cut sites?
a. a haplotype consisting of a single fragment 5.7 kb in length
vs. a haplotype consiting of fragments 3.0 and 2.7 kb in length.
a. What is an annonymous probe and why is it so named?
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