Genetics: From Genes to Genomes Third Edition Powerpoint to accompany Genetics: From Genes to Genomes Third Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres Chapter 11 Prepared by Malcolm Schug University of North Carolina Greensboro Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

The Direct Detection of Genotype Distinguishes Individual Genomes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Outline of Chapter 11 Four classes of DNA variation for direct detection of genotype Protocols that use hybridization, electrophoresis, PCR, microarrays, sequencing Positional cloning How to examine genes behind complex inheritance Haplotype association studies to map disease loci Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Members of the same species show enormous sequence variation in their genomes. 1 in 1000 bp differ in any two randomly chosen humans. Since haploid genome is 3  109 bp, 3 million differences between any two randomly chosen individuals Most differences are in non-coding, nonregulatory regions. Alleles – any variations in the genome at a particular location (locus) Polymorphic – two or more alleles at a locus Polymorphism – the particular variation DNA marker – polymorphic locus useful for mapping studies, disease diagnosis Anonymous locus – position on genome with no known function Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Four classes of DNA polymorphisms Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Single nucleotide polymorphism (SNP) Single base-pair substitutions Arise by mutagenic chemicals or mistakes in replication Biallelic – only two alleles Ratio of alleles ranges from 1:100 to 50:50. 2001 – over 5 million human SNPs identified Most occur at anonymous loci. Mutation rate of 1 X 10-9 per locus per generation Very few are thus new mutation in the species. Useful as DNA markers Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Microsatellites 1 every 30,000 bp Repeated units 2 – 5 bp in length Mutate by replication error Mutation rate of 10-3 per locus per gamete Useful as highly polymorphic DNA markers Figure 11.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.3

Highly polymorphic minisatellites are generated by unequal crossing-over. Figure 11.4 Repeating units 20-100 bp long Total length of 0.5 – 20 kb 1 per 100,000 bp, or about 30,000 in whole genome Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.4

Deletions, duplications, and insertions Expand or contract the length of nonrepetitive DNA Small deletions and duplications arise by unequal crossing over Small insertions can also be caused by transposable elements Much less common than other polymorphisms Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Deletions, duplications, and insertions Figure 11.5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Figure 11.5

SNP detection using southern blots Restriction fragment length polymorphisms (RFLPs) are size changes in fragments due to the loss or gain of a restriction site. Figure 11.6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.6

Must have sequence on either side of polymorphism SNP detection by PCR Must have sequence on either side of polymorphism Amplify fragment Expose to restriction enzyme Gel electrophoresis e.g., sickle-cell genotyping with a PCR based protocol Figure 11.7 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.7

SNP detection by ASO Figure 11.8 Very short probes (<21 bp) specific which hybridize to one allele or other Such probes are allele-specific oligonucleotides (ASOs). Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.8

ASOs can determine genotype at any SNP locus. Figure 11.9 a-c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.9 a-c

Hybridized and labeled with ASO for allele 1 Figure 11.9 d,e Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.9 d, e

Large-scale multiplex ASO analysis with microarrays can detect BRCA1 mutations. Figure 11.10 Each column contains an ASO differing only at the nucleotide position under analysis. BRCA1 DNA from any one allele can only be one of four ASOs in a column. Heterozygotes are easily detected. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.10

Primer extension to detect SNPs Figure 11.11 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Figure 11.11

Microsatellite allele detection analysis of size differences Figure 11.12 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.12

Huntington’s disease is an example of a microsatellite triplet repeat in a coding region. Figure 11.13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.13

Minisatellite detection and DNA fingerprinting 1985 – Alec Jeffreys made two key findings. Each minisatellite locus is highly polymorphic. Most minisatellites occur at multiple sites around the genome. DNA fingerprint – pattern of simultaneous genotypes at a group of unlinked loci Use restriction enzymes and southern blots to detect length differences at minisatellite loci. Most useful minisatellites have 10 – 20 sites around genome and can be analyzed on one gel. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Minisatellite analysis Figure 11.14 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.14

DNA fingerprints can identify individuals and determine parentage. e.g., DNA fingerprints confirmed Dolly the sheep was cloned from an adult udder cell. Donor udder (U), cell culture from udder (C), Dolly’s blood cell DNA (D), and control sheep 1-12 Figure 11.15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.15

Positional Cloning – use of polymorphic DNA markers to clone genes via linkage A pedigree of the royal family descended from Queen Victoria In which hemophilia A is segregating Figure 11.16 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.16 a

Blood-clotting cascade in which vessel damage causes a cascade of inactive factors to be converted to active factors Figure 11.16 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.16 b

Blood tests determine if active form of each factor in the cascade is present. Figure 11.16 c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.16 c

Techniques used to purify Factor VIII and clone the gene Figure 11.16 d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.16 d

Positional Cloning – Step 1 Find extended families in which disease is segregating. Use panel of polymorphic markers spaced at 10 cM intervals across all chromosomes. 300 markers total Determine genotype for all individuals in families for each DNA marker. Look for linkage between a marker and disease phenotype. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Once region of chromosome is identified, a high resolution mapping is performed with additional markers to narrow down region where gene may lie. Figure 11.17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.17

Positional cloning – Step 2 identifying candidate genes Once region of chromosome has been narrowed down by linkage analysis to 1000 kb or less, all genes within are identified. Candidate genes Usually about 17 genes per 1000 kb fragment Identify coding regions Computational analysis to identify conserved sequences between species Computational analysis to identify exon-like sequences by looking for codon usage, ORFs, and splice sites Appearance on one or more EST clones derived from cDNA Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Computational analysis of genomic sequences to identify candidate genes Figure 11.19 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.19

Gene expression patterns can pinpoint candidate genes. Look in public database of EST sequences representing certain tissues. Northern blot RNA transcripts in the cells of a particular tissue (e.g., with disease) separated by electrophoresis and probed with candidate gene sequence Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Northern blot example showing SRY candidate for testes determining factor is expressed in testes, but not lung, ovary, or kidney. Figure 11.20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.20

Positional cloning – Step 3 Find the gene responsible for the phenotype. Expression patterns RNA expression assayed by Northern blot or PCR amplification of cDNA with primers specific to candidate transcript Look for misexpression (no expression, underexpression, overexpression). Sequence differences Missense mutations identified by sequencing coding region of candidate gene from normal and abnormal individuals Transgenic modification of phenotype Insert the mutant gene into a model organism. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Transgenic analysis can prove candidate gene is disease locus. Figure 11.21 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.21

Example: Positional Cloning of Cystic Fibrosis Gene Linkage analysis places CF on chromosome 7 Figure 11.22 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.22 a

Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas. Figure 11.22 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.22 b

Location and number of mutations indicated under diagram of chromosome Every CF patient has a mutated allele of the DFTR gene on both chromosome 7 homologs. Figure 11.22 c Location and number of mutations indicated under diagram of chromosome Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.22 c

CFTR is a membrane protein. TMD-1 and TMD-2 are transmembrane domains. Figure 11.22 d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.22 d

Proving CFTR is the right gene Phenotype eliminates gene function. Cannot use transgenic technology Instead perform CRFT gene “knockout” in mouse to examine phenotype without CRFT gene. Targeted mutagenesis Introduce mutant CFTR into mouse embryonic cells in culture. Rare double recombinant events with homologous wild-type CFTR gene are selected for. Mutant cell is introduced into normal mouse embryos where they incorporate into germ line. Knockout mouse created Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Genetic dissection of complex traits Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

No environmental factor associated with likelihood of breast cancer Incomplete penetrance – when a mutant genotype does not always cause a mutant phenotype No environmental factor associated with likelihood of breast cancer Positional cloning identified BRAC1 as one gene causing breast cancer. Only 66% of women who carry BRAC1 mutation develop breast cancer by age 55. Incomplete penetrance hampers linkage mapping and positional cloning. Solution – exclude all non-disease individuals from analysis Requires many more families for study Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Variable expressivity Expression of a mutant trait differs from person to person. Include any degree of mutant phenotype as evidence for presence of mutant allele. Phenocopy Disease phenotype is not caused by any inherited predisposing mutation. Decreases power to detect correlation between inheritance of disease locus and expression of the disease Genetic heterogeneity Mutations at more than one locus cause same phenotype. Multiple families used in most studies If different families have different gene mutations, power of statistic to detect linkage will drop significantly. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Polygenic inheritance Two or more genes interact in the expression of phenotype. QTLs, or quantitative trait loci Unlimited number of transmission patterns for QTLs Discrete traits – penetrance may increase with number of mutant loci Expressivity may vary with number of loci. etc. Many other factors complicate analysis. Some mutant genes may have large effect. Mutations at some loci may be recessive while others are dominant or codominant. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Identifying contributing loci for complex traits Analyze two contrasting phenotypes from two inbred mouse lines. Eliminates heterogeneity Only two alternative alleles at every marker No environmental differences Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Pulp content in tomato – example of complex trait method Identification of two inbred strains with extreme, reproducible differences in pulp content Cross plants from high- and low-pulp strains. Cross identical F1 hybrids to generate several hundred F2 offspring. Range of phenotypes produced Figure 11.24 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.24 b

Identifying regions of chromosome where pulp content genes may lie Determine genotype at polymorphic DNA markers spaced at 20 cM intervals in each F2 plant. Look for correlation between marker genotype and pulp phenotype. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Haplotype association studies for high-resolution mapping in humans Haplotypes are sets of closely linked alleles. Specific combination of two or more DNA marker alleles situated close together on the same DNA molecule Usually SNPs Distance between SNPs on a haplotype must be short enough that they stay associated during transmission over many generations. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Formation of haplotypes over evolutionary time  Figure 11.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Ancient disease loci are associated with haplotypes. Start with population genetically isolated for a long time such as Icelanders or Amish. Collect DNA samples from subgroup with disease. Also collect from equal number of people without disease. Genotype each individual in subgroups for haplotypes throughout entire genome. Look for association between haplotype and disease phenotype. Association represents linkage disequilibrium. If successful, it provides high resolution to narrow parts of chromosomes. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Haplotype analysis provides high resolution gene mapping. Figure 11.26 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 11.26