Page 102 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

396 Chapter 11 Analyzing Genomic Variation

13. You want to make a recombinant DNA in which a DNA polymerase originally used for PCR, from
PCR product amplified from the human genome is in- the bacterium Thermus aquaticus, lacks the 3′-to-
serted into a plasmid vector. The polylinker of this 5′ exonuclease found in other DNA polymerases
vector includes recognition sites for the enzymes such as that from E. coli (review Fig. 7.9). Why do
EcoRI (5′ G^AATTC 3′) and BamHI (5′ G^GATCC scientists now most often use DNA polymerase
3′). (The ^ symbolizes the cut site in the DNA.) PCR from a different thermophilic bacterium
primers that could amplify the fragment of human (Pyrococcus furiosa) that does contain this exonu-
DNA are: 5′ GCTACTTCGCGTATTCCA 3′ and clease function?
5′ CCCAAGTCCTAGCCGATA 3′. 15. Problem 8 shows three different sequences of the
a. Describe in detail how these primers would need to same autosome in human populations. These se-
be modified to create a fragment of the human ge- quences are each from a single chromatid. You know
nome flanked by EcoRI sticky ends so that this this to be true because the PCR amplifications were
fragment could be cloned easily into the plasmid from individual haploid gametes. If you wanted to ob-
vector. You will need to consider the fact that most tain the same information by PCR amplification of
restriction enzymes, including EcoRI, cannot cut genomic DNA from somatic cells, the problem would
DNA if the restriction site is directly at the end of be somewhat more complicated because the starting
the DNA molecule; the restriction enzyme recogni- cells are diploid. Each PCR product to be sequenced
tion site must be at least six base pairs distant from would thus actually be amplified from two homolo-
the end. gous chromosomes. You could still verify the exis-
b. Describe a potential feature of the PCR-amplified tence of the three different haploid sequences shown
region of the human genome that could prevent you in Problem 8 by analyzing the somatic genomic DNA
from using the strategy you described in part (a). from as few as two people (if they happened to be the
c. Now describe how the primers must be modified right people).
to create a human DNA fragment with an EcoRI- a. Indicate the diploid genotypes of two people from
compatible single-stranded overhang at one end whom you could identify these three different hap-
and a BamHI-compatible overhang at the other loid sequences. Account for all 10 nucleotides in
end. (Two possibilities exist; you need to the sequences. Three possible correct answers ex-
describe only one. Assume that a BamHI site also ist; you need to show only one.
must be at least six base pairs from the end of b. If you PCR amplified DNA from somatic genomic
the DNA.) Why might you want to make such DNA from a person with one particular genotype,
a fragment? you would not be able to conclude that their ge-
14. You sequence a PCR product amplified from a per- nome contains any of these three sequences. What
son’s genome, and you see a double peak such as that is the genotype of this person? Explain why you
seen in Fig. 11.11b. Most of the time, this result indi- could not reach this conclusion.
cates that the person is a heterozygote for a SNP at 16. The trinucleotide repeat region of the Huntington dis-
that position. But it is also possible that the result is ease locus (HD) in six individuals is amplified by
due to a mistake in DNA replication during the PCR PCR and analyzed by gel electrophoresis as shown in
amplification, with DNA polymerase misincorporat- the following figure; the numbers to the right indicate
ing the wrong nucleotide. the sizes of the PCR products in bp. Each person
a. If you saw an artifactual double peak in the sequence whose DNA was analyzed has one affected parent.
trace, did the mistake happen in the first few rounds a. Which individuals are most likely to be affected by
of PCR amplification or in the last few rounds? Huntington disease, and in which of these people is
b. Whether or not you see a double peak, is it more the onset of the disease likely to be earliest?
likely that a mistake would happen in the first b. Which individuals are least likely to be affected by
few rounds of PCR amplification or in the last the disease?
few rounds? c. Consider the two PCR primers used to amplify the
c. Given that mistakes can happen during PCR ampli- trinucleotide repeat region. If the 5′ end of one of
fication, what could you do to be sure of a person’s these primers is located 70 nucleotides upstream of
genotype? Why would this degree of certainty be the first CAG repeat, what is the maximum dis-
difficult to achieve if you were doing preimplanta- tance downstream of the last CAG repeat at which
tion genotyping of embryos? the 5′ end of the other primer could be
d. PCR relies on heat-stable DNA polymerases from found? [Assume that the diagram shows the largest
+
thermophilic bacteria that grow in hot springs. The HD allele possible (that is, 35 CAG repeats).]