Page 98 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

392 Chapter 11 Analyzing Genomic Variation

WHAT’S NEXT

In this chapter and in Chapters 9 and 10, we have focused different types of proteins that help package and manage the
on the nucleotide content of genomes, particularly the information carried by DNA. These proteins have many
6 billion nucleotides organized into 46 chromosomes in roles. Certain proteins help compact the chromosomes to fit
each normal human diploid cell. In the next several chap- in the nucleus. Some proteins ensure that the chromosomal
ters, we examine features of the chromosomes that allow DNA is properly duplicated during each cell cycle, while
these DNA sequences to function properly and to be trans- others govern the distribution of chromosomes to daughter
mitted from one generation to the next. cells. Yet other proteins are responsible for regulating the
We begin by considering how in spite of the enormous availability of genes to the transcriptional machinery so that
complexity of DNA sequences, the DNA actually constitutes the genes can be expressed into proteins. In Chapter 12, we
only about one-third of the total mass of a chromosome. The examine how proteins interact with DNA to generate the
remainder of the chromosome is made of thousands of functional complexity of a chromosome.

SOLVED PROBLEMS

I. Genomic DNA from a woman’s blood cells is PCR maternally derived chromosome and one on the paternally
amplified by a single pair of primers representing a derived chromosome), as long as the primer can hybridize to
unique locus in the genome. The PCR products are both homologs as is usually the case. The DNA sequence
then sequenced by the Sanger method, using one of the trace has two nucleotides at several positions. This fact indi-
PCR primers as a sequencing primer. The following cates that the woman must be a heterozygote and that the
figure shows a trace of just part of the sequence read. PCR is amplifying both alleles of the locus.
a. Notice that both alleles contain multiple repeats of
G T A C
the dinucleotide CA. The most likely explanation
for the polymorphism is therefore that the locus
contains an SSR polymorphism whose alleles have
different numbers of CA repeats. One allele has
six repeats; the second allele must have more
CA units.

Smaller Larger b. Writing out the first 14 nucleotides of both alleles is
straightforward. If the assumption in part (a) is cor-
a. What kind of polymorphism is most likely represented? rect, then one allele should have more than six CA
b. With your answer to part (a) in mind, determine the repeats. The trace shows evidence for two additional
woman’s genotype at this locus. Indicate all nucleo- CA repeats in one allele at positions 15–18, for a to-
tides that can be read from both alleles and their tal of eight CA repeats.
5′-to-3′ orientation. You can then determine the nucleotides beyond
c. What kind of molecular event was likely to have gen- the repeats in the shorter allele by subtracting CACA
erated this polymorphism? from positions 15–18. The remaining peaks at these
d. How would you know exactly where in the genome positions correspond to ATGT. Note that ATGT can
this locus is found? also be found in the longer allele, but now at nucleo-
e. What is another way in which you could analyze the tides 19–22, just past the two additional CACA re-
PCR products to genotype this locus? peats. You can determine the last four nucleotides in
f. Suppose you wanted to genotype this locus based on the shorter allele by subtracting ATGT from positions
single-molecule DNA sequencing of whole genomes 19–22, revealing TAGG. The sequences of the two
as shown in Fig. 9.24. Would a single read suffice for alleles of this SSR locus (indicating only one strand
genotyping the locus by this alternative method? of DNA each) are thus:
Allele 1: 5′...GGCACACACACACAATGTTAGG...3′
Answer Allele 2: 5′...GGCACACACACACACACAATGT...3′
To solve this problem, you need to understand that PCR will c. The mechanism thought to be responsible for most
simultaneously amplify both copies of a locus (one on the SSR polymorphisms is stuttering of DNA polymerase
DNA: © Design Pics/Bilderbuch RF during DNA replication.