Bios 320: Gene Technology Lab and Problems Fall 1997, Part I (Fornari)

1. Restriction Enzymes
2. DNA sequencing
3. Polymerase Chain Reaction

p. 456-460
p. 478-480
p. 482-487

# 15.10, 15.11, 15.12 from the Russell text, Ch. 15

and the following problems: (from the Griffith's et. al. text)

1. You have a purified DNA molecule, and you wish to map restriction-enzyme sites along its length. After digestion with EcoRI, you obtain 4 fragments numbered 1, 2, 3, and 4. After digestion of each of these fragments with HindIII, you find that fragment 3 yields two subfrag-ments (3-1 and 3-2) and that fragment 2 yields three subfragments (2-1, 2-2, 2-3). After diges-tion of the entire DNA molecule with HindIII, you recover four pieces: A, B, C, and D. When these pieces are treated with EcoRI, piece D yields fragments 1 and 3-1, A yields 3-2 and 2-1, and B yields 2-3 and 4. The C piece is identical with 2-2. Draw a restriction map of this DNA.

2. A linear fragment of DNA is cleaved with the individual restriction enzymes HindIII and SmaI and then with a combination of the two enzymes. The fragments obtained are:

HindIII 2.5 kb, 5.0 kb
SmaI 2.0 kb, 5.5 kb
HindIII, SmaI 2.5 kb, 3.0 kb, 2.0 kb

b. The mixture of fragments produced by the combined enzymes is cleaved with the enzyme EcoRI, resulting in the loss of the 3.0 kb fragment (band stained with ethidium bromide on an agarose gel) and the appearance of a band stained with ethidium bromide representing a 1.5 kb fragment. Mark the EcoRI cleavage site on the restriction map.

Bios 320: Gene Technology Lab and Problems Fall 1997, Part II

1. Mutations
2. Molecular Analysis of Sickle Cell Anemia
3. RFLP's

--what are they?
--how are they used for linkage analyses?

--Proportion of Polymorphic loci in a population
--Heterozygosity in individuals
--Heterozygosity at the nucleotide level; equation (see handout)

--what are they?
--how are they used in forensic analysis, and for linkage studies?

Ch.9 p.280-282
Ch.15 p.487-489, p.495-496
Ch.21 p.709-716

Assigned Problems:

#'s 15.13, 15.14, 15.16, 15.19
#'s 21.29, 21.30, 21.31

1. In mouse HindIII restriction digests, a certain probe picks up a simple RFLP consisting of two alternative alleles of 1.7 kb and 3.8 kb. A mouse heterozygous for a dominant allele for bent tail and also heterozygous for the above RFLP is mated to a wild-type mouse that shows only the 3.8 kb fragment. Forty percent of the bent-tail progeny are homozygous for the 3.8 kb allele, and 60 percent are heterozygous for the 3.8 and 1.7 kb forms.

a. Is the bent-tail locus linked to the RFLP locus? Draw the parental and progeny chromosomes to illustrate your answer.
b. What RFLP types do you predict among the wild-type offspring, and in what proportions?