Page 79 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

11.2 Genotyping a Known Disease-Causing Mutation 373

DNA polymerase to generate new strands of DNA comple- (CNVs) affect many nucleotide pairs that extend over
mentary to both strands of genomic DNA between the regions much larger than can be amplified into a PCR prod-
primers; remember that DNA polymerase adds nucleotides uct, so they must be analyzed by other methods that will be
sequentially onto the 3′ end of a primer. described later in the book.
After sufficient time has elapsed to allow copying of
the target region, the reaction is heated in order to melt Sequencing PCR products
apart the original template strands of DNA from the newly As you may remember, the mutation Hbβ that causes
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synthesized strands. The reaction tube is allowed to cool, sickle-cell anemia changes the identity of a single amino
so that the starting DNA and the copies synthesized in the acid in the β chain of adult hemoglobin from glutamic acid
previous step become templates for further replication, us- to valine. The mutant allele is a single nucleotide substitu-
ing the oligonucleotides remaining in the tube as primers. tion that changes an A to a T in the mRNA-like strand of
Performing this same sequence of steps—denaturation into the β-globin gene; the mutation is thus a single nucleotide
single strands, hybridization of primers, and polymeriza- polymorphism (SNP) (Fig. 11.11a). By genotyping the al-
tion by DNA polymerase—as an iterative loop results in an leles of this SNP, we can identify people who will suffer
exponential increase in the number of copies of the target from sickle-cell anemia or are carriers for this trait.
region with each step (Fig. 11.10). Repeating the cycle just The process begins by PCR amplifying the locus from
22 times would generate more than a million double- the person’s genomic DNA using a pair of primers comple-
stranded copies of the target region; after 32 repetitions, the mentary to sequences on either side of the actual disease-
reaction tube would have over a billion copies of this part causing mutation (Fig. 11.11a). Once the PCR product is
of the genome. made, its DNA sequence can be determined by the auto-
The iterative steps of the protocol can be automated in
a PCR machine that heats up and cools down the sample mated Sanger method shown previously in Fig. 9.7. Either
one of the two PCR primers can serve as the primer for the
according to a preprogrammed schedule. The reaction sequencing reactions.
tubes placed into the machine contain enough nucleotide As Fig. 11.11b shows, the nucleotide substitution re-
triphosphates and oligonucleotide primers to support these sponsible for the disease shows up clearly in comparing the
multiple rounds of DNA replication. Moreover, the tubes sequence obtained from the PCR products in Hbβ Hbβ
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contain a special DNA polymerase from a bacterium that sickle-cell patients and Hbβ Hbβ normal homozygotes.
A
A
grows in hot springs. This DNA polymerase remains active But importantly, both alleles are visible simultaneously in
after being subjected to the high temperatures used to melt the sequence trace made from the PCR product generated
apart the DNA strands at each round of the PCR protocol. using Hbβ Hbβ heterozygous genomic DNA as the tem-
A
S
It is crucial for you to remember from Fig. 11.9 that the
two priming oligonucleotides dictate the nature of the final plate. Genomic DNA prepared from somatic cells of the
heterozygote contains both allelic variants. Because the
PCR product. The ultimate PCR product is a double- primers hybridize equally well with the two homologous
stranded fragment of DNA that extends from the position chromosomes (given that the sickle-cell mutation does not
of one primer’s 5′ end to the position of the other primer’s alter the genomic sequences complementary to the prim-
5′ end. The primers must be complementary to opposite ers), about half the DNA molecules in the final PCR prod-
strands and have 5′-to-3′ polarities that point toward each uct will contain the mutant sequence and the other half the
other through the region of interest. In practice, PCR is in- wild-type sequence. Heterozygosity for the disease- causing
efficient if the primers are far apart, so the protocol gener- SNP is thus seen as a double peak showing both A and T in
ally cannot amplify DNA regions greater than 25 kb long.
the DNA sequence trace.
The technique of sequencing PCR products amplified
from genomic DNA is a straightforward way to determine
PCR Products Are Genotyped one’s genotype for any SNP. The same method can also be
by Sequencing or Sizing used to genotype other kinds of polymorphisms involving
small numbers of nucleotides, such as small deletions/
For Mendelian genetic diseases caused by changes involv- insertions (DIPs) or expansions/contractions of the num-
ing only one or a few nucleotides in a single gene, all of the bers of repeats in simple sequence repeats (SSRs).
information that distinguishes a normal allele from a mu-
tant allele resides within a discrete region of the genome
that can be encompassed by a PCR product. The differ- Size variation in PCR products
ences between alleles can be recognized either by direct In some cases it is possible to genotype a polymorphism in
sequencing of PCR products, or in cases in which muta- a PCR product without actually sequencing it. Gel electro-
tions add or subtract nucleotide pairs from the genome, by phoresis can easily distinguish small variations in the actual
simply looking at the sizes of the PCR products. More size of a locus caused by DIPs or SSRs, as illustrated in
complex polymorphisms such as copy number variants Fig. 11.12. Again, you begin by using a pair of primers