2. Analysis of Genetic Variation I and II_McDermott_NOTES.pdf

Genetic Variation [1] ANALYSIS OF GENETIC VARIATION A. CHROMOSOMES AND CYTOGENETICS 1. Structure of Metaphase Chromosomes 2. Banding of Metaphase Chromosomes 3. Fluorescent In Situ Hybridization (FISH) B. KARYOTYPE ANALYSIS 1. Chromosome Number 2. Composition of Sex Chromosomes 3. Chromosome Struct...

Genetic Variation [1] ANALYSIS OF GENETIC VARIATION A. CHROMOSOMES AND CYTOGENETICS 1. Structure of Metaphase Chromosomes 2. Banding of Metaphase Chromosomes 3. Fluorescent In Situ Hybridization (FISH) B. KARYOTYPE ANALYSIS 1. Chromosome Number 2. Composition of Sex Chromosomes 3. Chromosome Structure C. PRINCIPLES OF GENETIC VARIATION 1. Allele 2. Polymorphism 3. Types of Polymorphisms 4. Linkage 5. Haplotype 6. Linkage Analysis D. ANALYSIS OF POLYMORPHISMS 1. Restriction Fragment Length Polymorphisms (RFLP) 2. PCR-based Methods for RFLP Analysis 3. DNA Microarray Analysis (SNP Chip) 4. Genome-Wide Association Studies (GWAS) E. GENETIC TESTS IN MEDICINE 1. Multiplex PCR 2. Allele-specific Oligonucleotide (ASO) Test F. GENETIC MAPPING 1. Genetic Map 2. Physical Map G. HUMAN GENOME PROJECT 1. Overview 2. Sequence Maps of Chromosomes Paul J. McDermott, Ph.D. Office: (843) 792-3462 [email protected] Genetic Variation [2] OBJECTIVES 1. Describe the structure of metacentric, submetacentric and acrocentric chromosomes. 2. Describe how chromosome banding is utilized for karyotype analysis. 3. Describe fluorescence in situ hybridization (FISH) and how is used for chromosome analysis. 4. Define a polymorphic allele. 5. Describe the 4 main types of polymorphisms in the human genome. 6. Describe linkage and how it effects the frequency of recombination between 2 or more genes on the same chromosome. 7. Define a haplotype. 8. Describe how polymorphisms such as restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) and variable number of tandem repeats (VNTRs) are used for linkage analysis. 9. Describe how DNA microarrays are used to detect single nucleotide polymorphisms (SNPs). 10. Describe how SNP microarrays are used for analysis of haplotypes, chromosomal insertions and deletions, and genome-wide association studies (GWAS). 11. Describe multiplex PCR and how it is used to detect genetic variations and mutations. 12. Describe the two versions of the allele-specific oligonucleotide (ASO) test and how these are used to detect known mutations. 13. Differentiate between a genetic map and a physical map of the genome. 14. Explain the difference between sequence-tagged sites (STS) and expressed sequence tags (EST). 15. Define a contig and describe how it is used to construct physical maps. Figures adapted from: • Medical Genetics: An Integrated Approach © 2017 McGraw-Hill Education. • Human Molecular Genetics © 1999, BIOS Scientific Publishers Ltd • Elsevier’s Integrated Genetics © 2007 by Mosby, Inc. • The Cell: A Molecular Approach © 2007, ASM Press and Sinauer Associates, Inc. Genetic Variation [3] A. CHROMOSOMES AND CYTOGENETICS 1. Structure of Metaphase Chromosomes Homologous Chromosomes Sister Sister Chromatids Chromatids Telomere Centromere Telomere Metacentric short arm “p ” long arm “q ” Submetacentric Acrocentric 2. Banding of Metaphase Chromosomes Metaphase chromosomes can be visualized by light microscopy using Giemsa stain (G-banding) and by fluorescent microscopy using quinacrine stains (Q-banding). In these staining procedures, each chromosome is characterized by a reproducible pattern of dark and light bands that are numbered from the centromere to the telomere. These patterns are used as the basis of karyotyping to identify chromosomal abnormalities. The molecular basis of chromosome banding is determined by factors such as base composition of DNA and extent of chromatin condensation. a) G-banding: A-T rich, heterochromatic regions of chromosomes stain darkly while G-C rich, euchromatic regions stain lightly. b) Q-banding: This fluorescent stain produces essentially the same banding pattern as Giemsa, but resolution of the bands is lower. c) R-banding: Before Giemsa staining, chromosomes are heated to melt the DNA double helix in the A-T rich regions that usually stain darkly. This creates a “reverse” or R-banding pattern because only the G-C rich regions take up the stain. R-banding is often used to examine regions of chromosomes that have high gene densities because they tend to be G-C rich. Human Male G-banding Genetic Variation [4] 3. Fluorescent In Situ Hybridization (FISH) FISH is a technique that utilizes fluorescent probes of DNA that are complementary to specific DNA sequences in the chromosomes. Hybridization of a probe to its complementary sequence in a chromosome can be visualized by fluorescence microscopy. FISH study demonstrating a 15q deletion in the Prader-Willi/Angelman syndrome region. Chromosome Painting: Modification of FISH procedure in which multiple fluorescent probes that are complementary to known sequences in each chromosome are used for hybridization. Probes specific to each chromosome emit light at a unique wavelength, thus, chromosomes in a spread can be identified by color and analyzed. B. KARYOTYPE ANALYSIS 1. Chromosome Number: Modal number of chromosomes (46 or 2n) 2. Composition of Sex Chromosomes: XX or XY 3. Chromosome Structure: Banding is used to describe a particular location on a chromosome as designated by the International System for Human Cytogenetic Nomenclature (ISCN) committee. Example: Chromosome # Arm Region # within arm Band # within region Subband 17 q 2 3 .2 C. PRINCIPLES OF GENETIC VARIATION Genetic Variation [5] 1. Allele: Variant form of a gene or DNA sequence at a specific locus in homologous chromosomes. 2. Polymorphism: Nucleotide sequence variation at a specific locus in a chromosome. An allele is considered polymorphic if two or more sequence variants are maintained at a frequency of at least 1% in the population. Polymorphism is not a mutation A a Individual Genotype = Aa Alleles A and a in this locus are polymorphic 3. Types of Polymorphisms • Polymorphisms are dispersed throughout the human genome. Greater than !⁄" of the protein coding loci in genes have sequence variants that are classified as polymorphisms. • Most polymorphisms are not actually located within gene sequences. a) Single Nucleotide Polymorphisms (SNPs): Sequence variation of a single bp. SNPs account for about 90% of human variation as approximately 15 million are dispersed in the genome. b) Simple Sequence Repeats (SSRs or Microsatellites): Highly polymorphic number of small repeats 1-10 bp in length. In general, SSRs are easy to analyze by PCR-based methods. c) Variable Number of Tandem Repeats (Minisatellites): Highly polymorphic number of repeats that are relatively larger (10-60 bp) than SSRs. d) Insertions or deletions of bases (INDELS) Second most abundant form of polymorphisms in humans. Most are classified as short, i.e. gain or loss of up to 50 bp. 4. Linkage: Two genes located on different chromosomes will segregate by the law of independent assortment. Thus, the combination of alleles in offspring follows the expected frequencies based on probability. For two genes on the same chromosome, the segregation of alleles in offspring may not be random if they are linked, that is, they are located close together. Linkage disequilibrium occurs when the assortment of inherited alleles deviates from the predicted frequencies. • Frequency of recombination between 2 loci on the same chromosome increases as a function of the distance between them. • Unit of distance = centiMorgans (cM) • 1 cM = 1% probability of crossover between two loci on a chromosome • Average of 150 cM per chromosome, 1 cM = approximately 1000 kilobases Genetic Variation [6] C. PRINCIPLES OF GENETIC VARIATION 5. Haplotype: Two or more alleles on the same chromosome that are inherited as a unit. The alleles are inherited together because of linkage, that is, the loci are close to each other on the chromosome. AB AB Haplotypes ab ab 6. Linkage Analysis: Linkage analysis is used to identify a polymorphic allele that co-segregates with a gene of interest located on the same chromosome. The polymorphism is utilized as a marker to track a suspected gene within a family pedigree without actually knowing the mutation. By definition, this polymorphism must co-segregate with the gene of interest and be present in affected family members. D. ANALYSIS OF POLYMORPHISMS 1. Restriction Fragment Length Polymorphisms (RFLP) Polymorphisms are detected by a change in the RFLP pattern. Alleles are determined by the sizes of bands as detected by either Southern blotting or PCR analysis. a) Single nucleotide polymorphism (SNP) that either creates or destroys a restriction endonuclease site at a specific locus in the chromosome. A: 5´-G-A-A-T-T-C-3´ a: 5´-G-A-A-T-T-T-3´ Example: C/T SNP changes EcoR1 site: 5´-G-A-A-T-T-C-3´ 600 bp EcoR1 400 bp EcoR1 EcoR1 Normal Gene A EcoR1 EcoR1 Disease Gene Homologous chromosomes a Probe Probe Probe 1 2 3 1 • Digest genomic DNA with EcoR1 2 • Separate DNA fragments according to size by agarose gel electrophoresis 3 • Detect RFLP fragments by Southern blot using probes complementary to the shaded DNA sequences 500 250 0 1000 500 250 0 DNA Fragment (bp) 1000 DNA Fragment (bp) DNA Fragment (bp) wells 1000 500 250 0 AA Aa aa AA Aa aa AA Aa aa • If SNP C/T is linked closely linked to the gene, it will be inherited as a unit. Individuals who have a disease allele (a) will be linked to T while the normal allele A is linked to C. Genetic Variation [7] D. ANALYSIS OF POLYMORPHISMS 1. Restriction Fragment Length Polymorphisms (RFLP) b) Variable Number of Tandem Repeats (VNTR or Minisatellites) Example: VNTR at chromosome locus BamH1 = repeat sequence of 25 bp Normal Gene EcoR1 A BamH1 EcoR1 Homologous Chromosomes Disease Gene a 1 • Digest genomic DNA with BamH1 & EcoR1 2 • Southern blotting and hybridization using probe complementary to sequence in fragment. 3 • Size of RFLP fragment is directly proportional to the number of tandem repeats. • In this example, larger number of repeats are linked to disease gene. # of Repeats in RFLP Probe 10 5 0 AA Aa aa 2. PCR-based Methods for RFLP Analysis a) Simple Sequence Repeats (SSRs or Microsatellites) Example: Amplify SSR (GTn) by PCR using primers (P1 and P2) that are complementary to the sequences that flank either side of the SSR. The PCR products are then separated according to size by gel electrophoresis. P1 A Normal Gene (GT)20 P2 P1 (GT)10 PCR Product (bp) a Disease Gene P2 • PCR • Gel Electrophoresis • SSRs are useful for linkage analysis because they are highly polymorphic and present throughout the genome. > 20,000 SSRs in the human genome 200 150 0 Homologous Chromosomes AA Aa aa Genetic Variation [8] D. ANALYSIS OF POLYMORPHISMS 2. PCR-based Methods for RFLP Analysis b) Variable Number of Tandem Repeats (VNTR or Minisatellites) Example: VNTR sequence at a specific chromosome locus P1 Normal Gene A P2 P1 Disease Gene Homologous Chromosomes a = repeat sequence of 25 bp P2 • PCR using primers (P1 and P2) specific for VNTR flanking sequences PCR Product (bp) • Separate DNA fragments according to size by agarose gel electrophoresis 500 • Recombination frequency between the gene and the VNTR is a function of the relative distance between them on the chromosome. 250 0 AA Aa aa Genetic Variation [9] D. ANALYSIS OF POLYMORPHISMS 3. DNA Microarray Analysis (SNP Chips) • This technique is used to analyze thousands of genes simultaneously. Small sequences of DNA are spotted or synthesized onto a solid support of glass or plastic that measures about 1.5 cm2. • The sequences, which consist of synthetic oligonucleotides, cDNAs or small fragments of genomic DNA, are arranged in a grid pattern on the microarray. • In most applications of this technique, samples of DNA or RNA are labeled with a fluorescent tag and hybridized to the microarray. A computer-based laser detection system is used to analyze the hybridization signals based on the fluorescence of each spot on the grid and to generate a pattern or “signature”. • Commercial human SNP microarrays have about 1.8 million genetic markers on the chip, which includes >900,000 SNPs and >950,000 probes for detecting variations in copy number. a) Overview of SNP Microarray Method Restriction Enzyme Digestion Ligation of Adapter DNA Genomic DNA = restriction endonuclease sites One primer PCR for random amplification of sequences in range of 250 – 1000 bp Hybridization Laser detection of signals and analysis of data Each spot on microarray contains a unique sequence (probe) specific for SNP In this schematic, the region of the microarray in the upper right corner is magnified to show 16 spots, each one containing a unique DNA sequence or probe. A probe will hybridize to complementary sequences of fluorescently-labeled DNA fragments derived from samples of genomic DNA prepared as shown above. If hybridization occurs, then the corresponding spot on the microarray will give a signal as indicated. Fragmentation and Labeling 50 – 100 bp 250 - 1000 bp Genetic Variation [10] D. ANALYSIS OF POLYMORPHISMS 3. DNA Microarray Analysis (SNP Chips) b) Analysis of Single Nucleotide Polymorphisms (SNP) Approximately 15,000,000 SNPs are dispersed in the human genome, accounting for about 90% of the sequence variation. The majority of SNPs (about 2/3) are C or T as shown below. Allele A • • • A-G-C-C-A-T-C-A-T-C-G-G-A-C-G-T-A-G-C-C-G-G • • • Allele B • • • A-G-C-C-A-T-C-A-T-T-G-G-A-C-G-T-A-G-C-C-G-G • • • c) Classification of SNPs • Linked SNPs: These are located in between genes in the spacer DNA. They have no effect on gene function or phenotype. SNPs have enormous potential in karyotyping individuals for genetic predisposition to certain diseases or responsiveness to therapeutic agents or drugs. • Causative SNPs: These are located in the regulatory region, splice site sequence or the coding sequence of a gene. Thus, SNPs can impact the phenotype of an individual by altering the structure/function of a protein or by changing its level of expression. d) Utility of SNP Microarray Data: Haplotypes • SNP microarrays produce a signature pattern and reveal haplotypes of closely linked SNPs inherited as a unit. A haplotype in a large group or cohort of individuals can be associated with disease status (affected or unaffected) or some other trait(s). • Statistical tests are used to determine if frequency of a genetic variant is different between samples and an odds ratio is calculated. In this example, 3 SNPs on 2 alleles can lead to 23 = 8 possible haplotypes. D. ANALYSIS OF POLYMORPHISMS 3. DNA Microarray Analysis (SNP Chip) Genetic Variation [11] e) Utility of SNP Microarray Data: Copy Number • SNP microarrays are used routinely to analyze chromosomes copy number, for example, chromosomal insertions and deletions. • SNP arrays can detect loss of heterozygosity (LOH) of a normal allele in which the other allele is either inactivated or abnormal. Example: Individual with large (>1 Mb) deletion and duplication in chromosome 20 Allele Frequency: If a deletion is is present, values cluster at 0 or 1 but are absent at 0.5. If a duplication is present, values cluster at at 0, 0.33, 0.67, and 1, reflecting the possible genotypes: AAA, AAB, ABB, BBB LogR Ratio: These values are a normalized measure of total signal intensity. If a deletion is present, values for SNP markers in the region decrease. If a duplication is present, the LogR values increase. 4. Genome-Wide Association Studies (GWAS) • Genome-wide association studies (GWAS) examine thousands of SNPs mapped throughout the entire human genome to look for linkages between SNPs and disease status in a cohort of individuals. This approach can help to identify a specific chromosome region or locus that might be associated with a disease or other trait(s). • Below is an example of a Manhattan plot depicting several strongly associated SNPs from a GWAS study. Four novel loci (19q13, 6q24, 12q24, and 5q14) were identified. Genetic Variation [12] E. GENETIC TESTS IN MEDICINE 1. Multiplex PCR • Multiplexing is a PCR reaction in which multiple target sites in a region of DNA are amplified simultaneously by adding multiple primer sets. Primer sets must be carefully designed and optimized to ensure compatibility under a common set of PCR reaction conditions. • Primers sets are labeled with a fluorescent tags of different colors, which is incorporated into the PCR products. • Primers are designed to generate a different size fragment for each DNA target sequence. • The labeled PCR products are separated by size using a capillary gel electrophoresis system and a laser detector is used to distinguish between different colors. Gene Segment Capillary Gel Blue Blue Green Fragment size Green Red Red 1000 bp 500 2000 bp 1000 3000 bp 1500 2. Allele-specific Oligonucleotide (ASO) Test a) Dot Blot Method: ASO probes are designed to detect a known mutation by hybridization. Normal DNA DNA from Sickle Cell Anemia Patient (SCA) Hybridize with complementary ASO probes Normal DNA Carrier SCA DNA Normal Allele Mutant Allele Normal DNA Carrier SCA DNA Genetic Variation [13] E. GENETIC TESTS IN MEDICINE 2. Allele-specific Oligonucleotide (ASO) Test b) Reverse Dot Blot Method • A sample of patient DNA is used for a multiplex PCR reaction with a label incorporated into the products. A strip of nylon membrane has probes for normal sequences and known mutated sequences along the length of the gene segment. Each shaded line on the strip contains a different ASO probe complementary to either a normal or mutated sequence. ASO probe Labeled Multiplex PCR products Hybridization + • The sample of labeled DNA fragments generated by the multiplex PCR reaction is hybridized to the strip. DNA fragments will hybridize to either normal or mutant ASO probes on the strip if the complementary sequence has been amplified from the sample. Normal Hybridize Mutated sequence probes Labeled Multiplex PCR products Normal sequence probes Heterozygous (carrier) Homozygous Genetic Variation [14] F. GENETIC MAPPING 1. Genetic Map: A map of relative positions of genes and markers on a chromosome as determined by recombination frequency of linked alleles. The distances are measured in centiMorgans (cM). • Genetic maps are reliable for ordering of markers along chromosomes • Frequency of recombination is higher toward telomeres and in females as compared to males. Genetic maps do not always indicate actual distances between markers. • Genetic maps serve as a framework for more detailed physical maps. 2. Physical Map: A map of actual physical distances between genes and other markers on a chromosome as measured in base pairs. a) Sequence-tagged Site (STS): Unique DNA sequence in the genome whose exact location and sequence are known, ranging between 200-500 bp in length. STS databases are used as landmarks in physical maps of the genome. STS can be detected by PCR. Overlapping clones in a genomic library STS1 STS2 Detect STS site in genomic clone by PCR amplification with primers specific for the STS sequence STS3 5´ 3´ 3´ 200-500 bp 5´ Genetic Variation [15] F. GENETIC MAPPING 2. Physical Map b) Expressed Sequence Tags (EST): Short sequences of cDNA derived from mRNA of a given cell type, ranging between 200-500 bp in length. ESTs represent expressed portions of a gene and are mapped to specific locations in the chromosomes. EST databases are used to search for sequence similarities in unknown genes. c) Assembly of Overlapping Clones (Contigs): A series of overlapping DNA sequences used to make a physical map that reconstructs the original DNA sequence of a chromosome or a region of a chromosome. E F D A E B C Fragments from genomic library B C with markers A B B C C D Align markers to produce contigs E E F G. HUMAN GENOME PROJECT 1. Overview 2. Sequence Maps of Chromosomes: Genetic markers and DNA sequence between markers http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid=9606