Lecture 26: RFLPs, DNA fingerprinting, polymerase chain reaction

Please note that some of these questions include material from earlier lectures.

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?

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.

a. How many different haplotypes would be seen in a survey of a population that includes polymorphisms at both cut 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 polymorphisms might be obtained with probe Z and appropriate restriction endonucleases with six nucleotide recognition 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.

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 (see boxed example 13.5 in the textbook if you do not remember this).

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. (We have not dealt with this in class. You will need to do some projection. Perhaps the best way to look at is to ask if you were the child or young adult, would you want to know?)

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. An "annonymous" probe designated G8 played an important role in the original identification of the genetic locus responsible for Huntington disease.

a. What is an annonymous probe and why is it so named?

b. What is the advantage of using annonymous probes rather than known genes?

c. The annonymous probe (G8) used in linkage studies on Huntington disease detected two closely linked polymorphic HindIII sites. What special advantage did this provide for the linkage studies?

d. Would you expect the same G8 haplotype to be associated with Huntington disease in all disease-prone families? Explain your answer.

e. Describe a DNA fingerprinting technique that would be more useful than the G8 probe for identifying children of Huntington disease patients who have inherited the disease allele, based on our current knowledge of the genetics of the disease. Also explain why you consider your proposed alternative assay to be superior.

19. What does each of the following acronyms stand for? Also, how is each of them used in genetic analysis or the study of genetics?

a. VNTR
b. RFLP
c. RAPD
d. STRP
e. PCR

20. Briefly summarize the techniques that are shared in common in the analysis of RFLP and VNTR patterns.

21. What is the principal difference between RFLPs and VNTRs?

22. Explain why VNTRs are never dominant as genetic markers.

23. DNA fingerprinting has become a widely used technique.

a. What is meant by DNA fingerprinting?

b. What types of markers are most commonly used for DNA fingerprinting?

c. Can this technique be used for species other than humans? Explain your answer.

d. Why is DNA fingerprinting frequently done on a DNA sample that has first been amplified by PCR?

e. How many different haplotypes do you expect the DNA fingerprint for an individual to contain for each marker examined?

f. How would your answer differ in part e if the sample has been amplified by PCR from a single sperm cell?

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