Page 80 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

374 Chapter 11 Analyzing Genomic Variation

Figure 11.11 Detection of the sickle-cell mutation by Figure 11.12 Detection of simple sequence repeat (SSR)
sequencing of PCR products. (a) The mutation responsible for polymorphisms by electrophoresis of PCR products.
sickle-cell anemia is a SNP that changes a single amino acid in the (a) SSR alleles differ in length. Left and right primers correspond to
hemoglobin β polypeptide from glutamic acid (E) to valine (V). This unique sequences that flank the SSR locus. (b) Genomic DNA is
polymorphism is PCR amplified by primers complementary to amplified by PCR with primers specific for the particular SSR locus.
(c) Gel electrophoresis and ethidium bromide staining distinguish
flanking sequences that do not vary between alleles. (b) Sequencing (a) Synthesize primers corresponding to sequences flanking
the alleles from each other. (d) SSRs are highly polymorphic with
of PCR products made from genomic DNA templates. Note that the repeat locus.
many different alleles present in a population, but each person has
sequence of the PCR product from a heterozygous carrier shows (a) Synthesize primers corresponding to sequences flanking
Left primer
S
A
both the normal (Hbβ ) and mutant (Hbβ ) nucleotides at the repeat locus. Allele 1
only two alleles of any given SSR locus.
(a) Synthesize primers corresponding to sequences flanking
3'
5'
Left primer
position of the substitution (black arrow). repeat locus.
(a) Synthesize primers corresponding to sequences flanking 3'
5'
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Allele 1
3'
5'
Left primer
Courtesy of Joshua J. Filter, Cornell University, Ithaca, New York repeat locus. GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 5'
3'
(a) PCR amplifying alleles of the Hb gene 5' 5' 3' CA CA CA CA CA CA CA CA CA 3' 5' 3'
Allele 1 CA CA CA CA CA CA
(a) PCR amplifying alleles of the Hb gene
Left primer
5' 3' 5' 3' GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 3' 5'
Right primer
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Allele 1
3'
5'
3'
Hb A A Left primer GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 3' 5' 5'
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Hb
Right primer
Allele 2
5' 3'3' GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 3' 5' 5'
Left primer Right primer
Primer 1 5' CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA 3' CA CA 5' 3'
Primer 1
3'
5'
Allele 2
Right primer
4 4 5 5 6 6 7 7 8 8 3' Left primer GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 5'
GT
5'
3' CA CA
T T P P E E E E K K 5' Left primer CA CA CA CA CA CA CA CA CA 3' 5' 3'
Allele 2 CA CA CA CA CA CA CA CA
5' 5' ACTCCTGAGGAGAAG 3' 3' 5' 3' 5' 3' CA CA GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 3' 5'
GT GT
ACTCCTGAGGAGAAG
Right primer
CA CA CA CA CA CA CA CA CA
Allele 2 CA CA CA CA CA CA CA CA
3' 3' TGAGGACTCCTCTTC 5' 5' 3' CA CA GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 3' 5' 5'
TGAGGACTCCTCTTC
GT GT
3'
5'
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Right primer
3' GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT 3' 5' 5'
3'
5'
Primer 2 (b) Amplify alleles by PCR. Right primer
Primer 2
Left primer Left primer Right primer
Hb S S (b) Amplify alleles by PCR. 5' 3' Allele 2
Hb
5'
3'
Allele 1
Left primer Left primer
(b) Amplify alleles by PCR.
3'
3'
5'
5'
Primer 1
(b) Amplify alleles by PCR. 5'
Primer 1 Left primerAllele 1 3' Left primer Allele 2 3' 5'
4 4 5 5 6 6 7 7 8 8 Left primer Allele 1 Right Left primer Allele 25' 3' Right
5'
3'
5'
5'
T T P P V V E E K K 3' primer 3' primer
5' 5' ACTCCTGTGGAGAAG 3' 3' 5' 3' Allele 1 3' Right 5' 3' Allele 2 3' Right 5'
ACTCCTGTGGAGAAG
5'
3' 3' TGAGGACACCTCTTC C 5' 5' primer primer
TGAGGACACCTCTT
Right
Right
5'
3' primer 3' primer
5'
Right Right
primer primer
Primer 2
Primer 2
(b) Genotyping for sickle-cell anemia
(b) Genotyping for sickle-cell anemia (c) Analyze PCR products by gel electrophoresis.
1 2
(c) Analyze PCR products by gel electrophoresis.
(c) Analyze PCR products by gel electrophoresis.
2
1
2
1
(c) Analyze PCR products by gel electrophoresis.
Direction of
Hb A A 1 2 electrophoresis
Hb
Homozygote
Homozygote Direction of
electrophoresis
Direction of
electrophoresis
A C T C C T G Direction of
A C T C C T G G G A G A A GA G G A G A A GA
electrophoresis
(d) Example of population with three alleles
Allele 1
A A
Hb / Hb
Hb / Hb S S (d) Example of population with three alleles
Heterozygote CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Heterozygote
Allele 1
(d) Example of population with three alleles
Allele 2
Allele 1 CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA
(d) Example of population with three alleles
A A
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Allele 1 CA CA CA CA CA CA CA CA
A C T C C T G G G A G A A G G
A C T C C T G G G A G A A CA CA CA CA CA CA CA CA CA CA CA
Allele 2
T T
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Allele 3
CA CA CA CA CA CA CA CA CA CA CA
Allele 2 CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CACACA
Allele 2
Allele 3
Hb
Hb S S CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
Allele 3 CA CA CA CA CA CA CA CA CA
CA CA CA CA CA CA CA CA CA
CA CACA
Homozygote Six diploid genotypes are present in this population.
Homozygote
Allele 3 CA CA CA CA CA CA CA CA CA
CA CA CACA CA CA CA CA CA CA CA CA CA
1/2
2/3
1/3
1/1
2/2
3/3
Six diploid genotypes are present in this population.
CA Allele CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA
CA CACA
T G G A G A A
A C T C C T G G G A G A A G G
A C T C C T G T Six diploid genotypes are present in this population.
3/3
1/2
2/2
1/1
1/3
2/3
3
Direction of
Allele
Six diploid genotypes are present in this population.
3/3
2/3
1/3
1/2
1/1
2/2
2
3
Allele electrophoresis
1 1/1 2/2 3/3 1/2 1/3 2/3 Direction of
Allele electrophoresis
3 2
Direction of
3 2 1 electrophoresis
Direction of
2 1 electrophoresis
1