Chromosomal Structure and Chromosomal Mutations Chapters 8 and 9 Objectives Define mutations and polymorphisms. Distinguish the three types of DNA mutations: genome, chromosomal, and gene. Diagram a human chromosome and label the centromere, q arm, p arm, and telomere. Illustrate the different types of structural mutations that occur in chromosomes. Show how karyotypes reveal chromosomal abnormalities. Describe interphase and metaphase FISH analyses. Mutations and Polymorphisms Mutation: a permanent transmissible change in the genetic material, usually in a single gene – Relatively small portion of population ( 1%) – “Variant” to describe inheritable trait, “mutation” to describe somatic changes Polymorphism: two or more genetically determined, proportionally represented phenotypes in the same population – Change in 1-2% of population – Mutations that do not severely affect phenotype (in general, exception is a balanced polymorphism) Balanced Polymorphism If natural selection eliminates individuals with detrimental phenotypes from a population, then why do harmful mutant alleles persist in a gene pool? A disease can remain prevalent when heterozygotes have some other advantage over individuals who have two copies of the wild type allele. When carriers have advantages that allow a detrimental allele to persist in a population, it creates a balanced polymorphism . Examples of Balanced Polymorphisms: Sickle Cell Disease Sickle cell carriers are resistant to malaria People who inherited one copy of the sickle cell allele had red blood cell membranes that did not admit the parasite. Carriers had more children and passed the protective allele to approximately half of them. Gradually, the frequency of the sickle cell allele in east africa rose from 0.1 percent to a spectacular 45 percent in thirty-five generations. Carriers paid the price for this genetic protection, whenever two produced a child with sickle cell disease. Glucose-6-Phosphate Dehydrogenase Deficiency G6PD deficiency is a sex-linked enzyme deficiency that affects 400 million people worldwide. It causes life-threatening hemolytic anemia which develops only under specific conditions- eating fava beans, inhaling certain types of pollen, taking certain drugs, or contracting certain infections. Studies on African children with severe malaria show that heterozygous females and hemizygous males for G6PD deficiency are underrepresented (enzyme deficiency gene somehow protects against malaria). Types of Mutations Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy) Chromosomal: abnormal chromosome structure Gene: DNA sequence changes in specific genes Chromosome Morphology Telomere: chromosome ends Centromere: site of spindle attachment – Constriction of the metaphase chromosome at the centromere defines two arms Nucleosome: DNA double helix wrapped around histone proteins Chromosome Facts Number of chromosomes: – 22 pairs + 1 pair sex-determining chromosomes = 46 – One chromosome of each pair donated from each parent’s egg or sperm – Sex chromosomes: X,Y for males; X,X for females – Largest chromosome #1 = ~263 million base pairs (bp) – Smallest chromosome Y = ~59 million bp Gene Facts Size of human genome: 3.4 billion base pairs (bp) Number of human genes: ~30,000 Genes vary in length and can cover thousands of bases – avg. size: ~3,000 bp Only about 5% of the human genome contains genes Function of much of the genome is unknown Human Genome Facts Gene dense “urban centers” (G+C rich) Gene poor “deserts” (A+T rich) Genes appear to be clustered in random areas with vast expanses of noncoding DNA between Stretches of up to 30,000 C-G bases often occur adjacent to gene rich areas, forming barrier between genes and “junk DNA” Human Vs Non-human Genome Organization Many other organisms’ Human genome has genomes are more greater proportion (50%) uniform, genes evenly of repeat sequences than spaced most other species Humans tend to produce – mustard weed (11%) more proteins due to – worm (7%) mRNA transcript – fly (35%) “alternative splicing” Humans stopped Humans have ~ same accumulating repetitive number of gene families DNA over 50 million years but many more members ago while other per family (especially organisms (mice) development, immunity) continue to accumulate repetitive DNA TYPES OF GENE ORGANIZATION Multigene families Pseudogenes – 5S RNA gene family – Conventional (stop (2000 copies of the codon mutants) same gene, – Processed (loss of chromosome 1) introns) – Globin gene family – Truncated (loss of (superfamily) sequences from one 7 globin genes, end) chromosome16 – Gene fragments 6 globin genes, chromosome 11 TYPES OF DNA: Nuclear Genomes Minichromosomes are short, rich in genes Macrochromosomes are larger but not necessarily rich in genes B chromosomes additional chromosomes possessed by some individuals in the population but not all CHICKEN NUCLEAR GENOME Total of 39 chromosomes 33 minichromosomes – Represents 33% total nuclear DNA – Contains 75% of the genes Six macrochromosomes – Represents 66% total nuclear DNA – Contains 25% of the genes B CHROMOSOMES Common in plants and also found in fungi, insects, and animals Appear to be fragmentary versions of normal chromosomes Some contain genes but isn’t clear whether they are active Can be affect phenotype (especially in plants where they reduce viability) CHROMOSOME NUMBERS FOR VARIOUS ORGANISMS Genome size, Number of Organism Mb chromosomes Saccharomyces 12.1 16 cerevisiae Drosophila 140 4 melanogaster Human 3,000 23 Maize 5,000 10 Salamander 90,000 12 The number of chromosomes refers to a haploid cell, which contains a single copy of the genome. SPECIAL FEATURES OF METAPHASE CHROMOSOMES Location of the Kinetochores centromere – Surface centromere – Centric, Acentric – Attachment of Karyogram microtubules – Banding patterns Telomere produced by specific – Mark end of staining techniques chromosome Alphoid DNA – Hundreds of repeating – 171 bp repeats at 5’TTAGGG3’ centromere Chromosome Morphology Telomere Short arm (p) Centromere Arm Long arm (q) Telomere Metacentric Submetacentric Acrocentric Defining Chromosomal Location Arm Region Band Subband 3 2 2 1 2 2 p 1 1 5 1 4 1 3 2 1 1 2 17q11.2 1 1 3 1 2 2 q 3 1 3 2, 3 4 1 2 2 4 3 Chromosome 17 Chromosome Morphology Changes During the Cell Division Cycle. DNA double helix: 2nm diameter Interphase (G1, S, G2) Chromatin “beads on a string:” 11nm Chromatin in nucleosomes: 30nm Metaphase (Mitosis) Extended metaphase chromosomes: 300 nm Condensed metaphase chromosomes: 700 nm Cell Division Cycle Interphase (11–30 nm fibers) G1 S G2 M Mitosis: Prophase Anaphase Metaphase Metaphase Telophase (300–700 nm fibers) Visualizing Metaphase Chromosomes Patient cells are incubated and divide in tissue culture. Phytohemagglutinin (PHA): stimulates cell division Colcemid: arrests cells in metaphase 3:1 Methanol: Acetic Acid: fixes metaphase chromosomes for staining Visualizing Metaphase Chromosomes (Banding) Giemsa-, reverse- or centromere-stained metaphase chromosomes G-Bands R-Bands C-Bands Karyotype International System for Human Cytogenetic Nomenclature (ISCN) – 46, XX – normal female – 46, XY – normal male G-banded chromosomes are identified by band pattern. Normal Female Karyotype (46, XX) (G Banding) Normal Female Karyotype (High-Resolution G Banding) Traditional Giemsa Banding of Chromosomes Chromosome Number Abnormality Aneuploidy (48, XXXX) Chromosome Number Abnormality Trisomy 21 (47, XX, +21) EPICANTHAL FOLD AND SIMIAN CREASE Down’s Syndrome And Maternal Age Age of Frequency of Down Mother Syndrome 30 1 in 800 35 1 in 384 36 1 in 307 37 1 in 242 38 1 in 189 39 1 in 146 40 1 in 112 45 1 in 32 TRISOMY 18 Congenital heart defects, multiple joint contractures, spina bifida, hearing loss, radial aplasia (underdevelopment or missing radial bone of forearm), cleft lip, birth defects of the eye TRISOMY 13 Congenital abnormalities include severe mental retardation, seizures, microcephaly, scalp defects (absent skin), microphthalmia, cleft lip and/or palate, hypotelorism, simian crease, extra digits TRISOMY 13 ANEUPLOIDY: TURNER’S SYNDROME, 45 XO Characterized by short stature and the lack of sexual development at puberty Klinefelter's Syndrome (XXY, Male) 1 in every 500 to 1000 male births. Small Firm Testicles Low Testosterone – Infertility – Incomplete Masculinization – Female Body Hair Distribution (Sparse facial, armpit, and pubic hair) – Decreased Libido 47,XYY Syndrome Causes no unusual physical features or medical problems Increased growth velocity during earliest childhood, with an average final height approximately 7 cm above expected final height Increased risk of learning difficulties (in up to 50%) and delayed speech and language skills Chromosome Structure Abnormalities Translocation Deletion Inversion Isochromosome Insertion Ring Derivative chromosome chromosome Translocations Chromosome Structure Abnormality: Balanced Translocation 45, XY, t(14q21q) Some Human Diseases Caused By Translocations Cancer – Several forms of cancer are caused by translocations – Described mainly in leukemia (AML and CML) Infertility – One of the would-be parents carries a balanced translocation, where the parent is asymptomatic but conceived fetuses are not viable. Down syndrome – A minority (5% or less) of cases due to a Robertsonian translocation of about a third of chromosome 21 onto chromosome 14 Philadelphia Chromosome Or Philadelphia Translocation A specific chromosomal abnormality that is associated with CML. Due to a reciprocal translocation designated as t(9;22)(q34;q11), which means an exchange of genetic material between region q34 of chromosome 9 and region q11 of chromosome 22. Translocation is a highly sensitive test for CML, since 95% of people with CML have this abnormality. Not sufficient to diagnose CML, since it is also found in ALL (25–30% in adult and 2–10% in pediatric cases) and occasionally in AML. Frequency Of Chromosomal Abnormalities Disorder Birth frequency Balanced translocation 1 in 500 Unbalanced translocation 1 in 2000 Pericentric inversion 1 in 100 Trisomy 21 1 in 700 Trisomy 18 1 in 3000 Trisomy 13 1 in 5000 47,XXY 1 in 1000 males 47,XYY 1 in 1000 males 47,XXX 1 in 1000 females Fluorescent in situ Hybridization (FISH) Hybridization of complementary gene- or region-specific fluorescent probes to chromosomes. Interphase or metaphase cells on slide (in situ) Probe Microscopic signal (interphase) Fluorescent in situ Hybridization (FISH) Metaphase FISH – Chromosome painting – Spectral karyotyping Interphase FISH FISH (Fluorescent In Situ Hybridization) Uses of Fluorescent in situ Hybridization (FISH) Identification and characterization of numerical and structural chromosome abnormalities. Detection of microscopically invisible deletions. Detection of sub-telomeric aberrations. Prenatal diagnosis of the common aneuploidies (interphase FISH). What is FISH used for? Locus specific probes – Hybridize to a particular region of a chromosome. Alphoid or centromeric repeat probes – Generated from repetitive sequences found at the centromeres of chromosomes – Because each chromosome can be painted in a different color, use this technique to determine whether an individual has the correct number of chromosomes or, for example, whether a person has an extra copy of a chromosome. What is FISH used for? Whole Chromosome Probes Collections of smaller probes, each of which hybridizes to a different sequence along the length of the same chromosome. Paint an entire chromosome and generate a spectral karyotype. Full color image of the chromosomes distinguishes between the chromosomes based on their colors, rather than based on their dark and light banding patterns. Whole chromosome probes are particularly useful for examining chromosomal abnormalities – For example, when a piece of one chromosome is attached to the end of another chromosome FISH Probes Chromosome-specific centromere probes (CEP) – Hybridize to centromere region – Detect aneuploidy in interphase and metaphase Chromosome painting probes (WCP) – Hybridize to whole chromosomes or regions – Characterize chromosomal structural changes in metaphase cells Unique DNA sequence probes (LSI) – Hybridize to unique DNA sequences – Detect gene rearrangements, deletions, and amplifications FISH Probes Telomere-specific probes (TEL) – Hybridize to sub telomeric regions – Detect sub telomeric deletions and rearrangements Probe binding site Telomere 100–200 kb 3–20 kb Unique sequences Telomere associated repeats (TTAGGG)n Genetic Abnormalities by Interphase FISH LSI Probe Greater or less than two signals per nucleus is considered abnormal. Cell nucleus Normal diploid signal Trisomy or insertion Monosomy or deletion Interphase FISH FISH Performed On Chromosomes From A Human PBL Chromosome 2: Green Chromosome 4: Orange Trisomy 21 FISH X chromosome: Green Y chromosome: Red Chromosome 21: Blue Microdeletion FISH Spectral Karyotyping Microdeletion Syndromes Currently Diagnosable with FISH Cri-du-Chat Miller-Dieker Syndrome Smith-Magenis Syndrome Steroid Sulfatase Deficiency DiGeorge/Velo-Cardio-Facial/CATCH- 22/Shprintzen Syndrome Kallman Syndrome Williams Syndrome Wolf-Hirschhorn Prader-Willi/Angelman Syndrome Cri-Du-Chat Syndrome Wolf-Hirschhorn syndrome (46,XY,del[4p]) Prader-Willi / Angelman Structural Abnormality by Interphase FISH LSI Probe (Fusion Probe) Structural Abnormality by Interphase FISH LSI Probe (Break Apart Probe) Translocation by Metaphase FISH WCP Probe (Whole-Chromosome Painting) TYPES OF DNA ORGANELLE GENOMES Most are circular, some linear (within the same organelle) Mitochondrial genomes – In humans: 10 copies/mitochondria, 800 mitochondria/cell – In S. cerevisiae: 100 copies/mitochondria, 65 mitochondria/cell Chloroplast genomes FEATURES OF MITOCHONDRIAL GENOMES FEATURE Homo sapiens S. cerevisiae Total no. of genes 37 35 Types of genes Protein coding genes 13 8 Ribosomal RNA genes 2 2 Transfer RNA genes 22 24 Other RNA genes 0 1 Number of introns 0 8 Genome size (kb) 17 75 ORGANIZATION OF mtDNA MAP OF MITOCHONDRIAL DNA WHERE DO YOU GET YOUR MITOCHONDRIA? Maternal inheritance only – Evolutionary biology – Mitochondria from same maternal lineage identical (forensic identification) Maternal and paternal inheritance – Heteroplasmy first observed in 1994 following identification of remains of the Romanovs The Romonovs: Czar Nicholas and Czarina Alexandra MITOCHONDRIAL DNA: Heteroplasmy Expected on some level in all individuals due to immense amounts of mtDNA May vary in different tissues (due to different mechanisms by which cells are generated) Sperm does carry mitochondria (100/sperm vs 100,000/oocyte) In mammals, 99.99% mtDNA is maternal – Paternal mtDNA diluted out – Implications for cloning whole organism MATERNAL INHERITANCE 18 DAUGHTERS OF A GENETIC EVE MITOCHONDRIAL DNA IN THE NEW WORLD 7 EUROPEAN DAUGHTERS OF EVE Helena Ursula – Clan lived in Pyrenees, migrated – Users of stone tools, drifted northward to England ~12,000 across all Europe yr ago Valda Jasmine – Originally from Spain (17.000 yr – Originate in Syria, decendents ago), migrated to Finland, traveled throughout Europe Norway Katrine Xenia – Lived in Venice 10,000 years – Caucasus Mountains 25,000 yr ago, most of clan live in the ago; just before Ice Age, clan Alps reached America Tara – Settled in Tuscany 17,000 yr ago, descendants northern Europe, the English Channel USES FOR mtDNA ANALYSIS Evolutionary relationships Molecular clocks Identification – Present in large quantities relative to nuclear DNA – Maternal relatives – Forensic analysis HUMAN GENOME PROJECT Medical Benefits Improved diagnosis of disease Earlier detection of predispositions to disease Rational drug design Gene therapy and control systems for drugs Pharmacogenomics “personal drugs” Organ replacement HUMAN GENOME PROJECT DNA Forensics Identify potential suspects at crime scenes Exonerate wrongly accused persons Identify crime and catastrophe victims Establish paternity and other family relations Identify endangered and protected species as an aid to wildlife officials (prosecution of poachers) HUMAN GENOME PROJECT DNA Forensics - cont. Detect bacteria and other organisms that may pollute air, water, soil, and food Match organ donors with recipients in transplant programs Determine pedigree for seed or livestock breeds Authenticate consumable such as caviar and wine HUMAN GENOME PROJECT Microbial Genome Research New energy sources (biofuels) Environmental monitoring to detect pollutants Protection from biological and chemical warfare Safe, efficient toxic waste cleanup HUMAN GENOME PROJECT Agriculture and Livestock Disease-, insect-, and drought-resistant crops More nutritious produce Biopesticides Edible vaccines incorporated into food products New environmental cleanup uses for plants like tobacco HUMAN GENOME PROJECT Evolution and Human Migration Use germline mutations in lineages to study evolution Study migration of different population groups based on female genetic inheritance Study mutations on the evolutionarily stable Y chromosome to trace lineage and migration Compare breakpoints in the evolution of mutations with ages of populations and historical events REPETITIVE DNA CONTENT Tandemly repeated DNA (Satellite DNA) Genomic DNA broken into 50-100 kb fragments is subjected to density gradient centrifugation – Main band (Buoyant density=1.701 g/cm3) – 3 Satellite bands (Buoyant density 1.687, 1.693, 1.697 g/cm3) REPETITIVE DNA CONTENT Minisatellites – Clusters up to 20kb, with repeat units up to 25 bp – Telomeric DNA Microsatellites – Shorter clusters, usually <150 bp, with repeat units of 4 bp or less – No two humans have exactly the same combination of microsatellite alleles (genetic profile) REPETITIVE DNA CONTENT: Interspersed Genome-Wide Repeats Transposition via RNA Transposition via DNA Retroelements DNA transposons – Retroviruses (RV) – Less than 1000/human – Endogenous RV genome – Retrotransposons – Horizontal gene transfer (McClintock’s jumping Human genes of maize) retroelements Insertion sequences – LINEs (long interspersed nuclear elements) (prokaryotic) – SINEs (short interspersed – Transposase nuclear elements): Alu, – Transposable phage ~106copies/ genome Gene Mutations Objectives Compare phenotypic consequences of point mutations. Distinguish detection of known mutations from scanning for unknown mutations. Discuss methods used to detect point mutations. Describe mutation nomenclature for expressing sequence changes at the DNA, RNA, and protein levels. Mutation Nomenclature 5162 G->A Replacement Base position Original base Deletion: 197delAG Insertion: 2552insT R197G Original aa Replacement aa position Point Mutations Gene mutations involving one or few base pairs. Not detectable by the cytogenetic method. Detected at the DNA sequence level. Point Mutations Do Not Always Have Phenotypic Effect. Type of DNA sequence Amino acid sequence mutation ATG CAG GTG ACC TCA GTG M Q V T S V None ATG CAG GTT ACC TCA GTG M Q V T S V Silent ATG CAA GTG ACC TCA GTG M Q L T S V Conservative Non- ATG CCG GTG ACC TCA GTG M P V T S V conservative ATG CAG GTG ACC TGA GTG M Q V T ter Nonsense ATG CAG GTG AAC CTC AGT G M Q V N L S Frameshift Types of Mutation Detection Methods Hybridization-based – SSCP, ASO, melt curves, array technology Sequencing (polymerization)-based – Sequence-specific PCR, allelic discrimination Cleavage-based – RFLP, nuclease cleavage, invader SEQUENCE SPECIFIC DETECTION METHODS Recognition of mutant sequences by restriction endonucleases – PCR-RFLP Recognition of sequence mismatch by DNA Polymerase – Allele specific amplification, DNA sequencing Recognition of sequence mismatch by physical hybridization – Dot blot, DNA microarray, melting point analysis Single-Strand Conformation Polymorphism Scans several-hundred base pairs. Based on intra-strand folding. – Single strands will fold based on sequence. – One base difference will affect folding. Folded single strands (conformers) can be resolved by size and shape. Strict temperature requirements. Single-Strand Conformation Polymorphism (SSCP) 1. Amplify region to be scanned using PCR. Normal control Test (with mutation) PCR products 2. Denature and dilute the PCR Single strands products. (conformers) 3. Separate conformers by PAGE or CGE. Single-Strand Conformation Polymorphism (SSCP) 4. Analyze results by comparison to reference normal control (+). PAGE CGE + + mut +/mut mut +/mut CGE=Capillary gel electrophoresis Single-Strand Conformation Polymorphism (SSCP) 5. Detect PAGE bands by silver staining. T1 T2 NC T1: test sample without mutation T2: test sample with mutation NC: normal control Allele-specific Oligomer Hybridization (ASO) Dot blot method Relies on binding effects of nucleotide mismatches. Specimen in solution is spotted on nitrocellulose. Labeled oligonucleotide probe is hybridized to immobilized specimen. Allele-specific Oligomer Hybridization (ASO) Three specimens spotted on duplicate membranes. One membrane exposed to probe complementary to the normal sequence (+ probe). One membrane exposed to probe complementary to the mutant sequence (m probe). m/+ +/+ m/m m/+ +/+ m/m + probe m probe Allele-specific Oligomer Hybridization (ASO) Chromogenic probe detection – 1 – normal (+/+) – 2 – heterozygous (m/+) – m – heterozygous mutant control – + – normal control – N – negative control 1 2 m + N 1 2 m + N + probe m probe SINGLE NUCLEOTIDE POLYMORPHISMS: SNPs Occur when a single nucleotide in the genome sequence is altered Two of every three SNPs involve CT Occur every 100-300 bases Occur in both coding (gene) and noncoding regions Many have no effect on cell function Human Genome Project and SNP Mapping Develop rapid, large scale identification and scoring of SNPs Identify common variants in the coding regions of most identified genes Create a SNP map of at least 100,000 markers Develop foundations for studies of sequence variation Variations And Mutations Identified ~1.4 million locations where SNPs occur in humans Ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females – Greater number of cell divisions required for sperm formation than for eggs Association analysis of SNPs in candidate disease genes and linkage disequilibrium mapping using SNPs SNP: Just Another Name for…Mutation SNP: Just Another Name for…Map Location SNP: Just Another Name for…Altered Expression SNP: Just Another Name for…Altered Regulation Melt Curve Analysis Number of repeat sequences present in a sample is determined by means of melting temperature analysis. Analysis of a target nucleic acid consisting of repetitive and non repetitive sequences comprising: – Hybridization of at least one hybridization probe comprising a first segment complementary to a non repetitive region and a second segment complementary to an adjacent repetitive region – Determination of the melting point temperature of the hybrid formed between the target nucleic acid and the at least one hybridization probe. Melt Curve Analysis Based on sequence effect on Tm. Can be performed with or without probes. Requires double-strand DNA–specific dyes. – Ethidium bromide – SyBr Green Also performed with fluorescence resonance energy transfer (FRET) probes. Melt Curve Analysis Double-stranded DNA specific dye (SyBr Green) will fluoresce when bound to DNA. Denaturation of DNA to single strands will result in loss of fluorescence. Fluorescence %SS DS=SS Tm %DS 50 Temperature (°C) 80 Melt Curve Analysis Every sequence has a characteristic Tm. Melt curve Tm indicates which sequence is present. Heterozygous (m/+) %SS DS=SS Homozygous normal (+/+) Homozygous mutant (m/m) %DS 50 Temperature (°C) 80 Melt Curve Analysis Detection instrument software may convert the melt curve to a derivative of fluorescence (speed of drop vs. temperature). Normal Heterozygous mutant Df/Dt Temperature (°C) Mutant Tm Normal Tm Melt Curve Melt Curve FRET: labels come into close proximity when their respective oligonucleotides adjacently anneal to the complementary target LED excites fluorescein, causes a transfer of energy and excitation of the LC Red 640 molecule. SNP detection by monitoring the temperature at which the longer wavelength fluorescent signal rapidly decreases indicating that one of the two probes has "melted" off of the template and that FRET is no longer occurring. Melting temperature of each sample reveals its classification as a wild type, mutant, or heterozygote. Array Technology Reverse dot blot methods. Used to investigate multiple genomic sites simultaneously. Unlabeled probes are bound to substrate. Specimen DNA is labeled and hybridized to immobilized probes. HLA DQ/HLA DQA1 System HLA: human lymphocyte antigens Determine whether patient has antibodies against a potential donor’s HLA antigens HLA DQ is historic name for this region A 242 bp region with variation detected with specific probes for subregions HLA DQ/HLA DQA1 System Original test could detect 6 common DQ alleles, 21 possible genotypes Subsequent analysis, renaming by geneticists resulted in DQA1 test, increased number of subtypes detected (up to 28 detectable types) Cheap, fast color reactions Reverse Dot Blot Reverse Dot Blot Analysis Commercially available strips containing oligonucleotides corresponding to specific genes/expressed sequence tags (ESTs) Sample is amplified by PCR and hybridized to strip Color detection system used to identify positive reaction between amplified DNA and test oligonucleotides PM plus DQA1 Typing Kit DQA1 TEST: Example 1 DQA1 TEST: Example 2 DQA1 TEST: Example 3 DQA1 TEST: Example 4 POLYMARKER (PM): PM plusDQA1 POLYMARKER (PM): PM plusDQA1 California Jogger: 1994 Drag marks found along the path near where the body was found Bloody clothes found along drag path, multiple tear and claw marks Scalping is consistent with mountain lion attack California Jogger: 1994 Mountain lions frequently return to the site of their prey Removed human victim, replace with carcass of a deer containing a tracking device When the tracking signal moved, brought in dogs, tracked and treed male mountain lion California Jogger: 1994 Was the animal captured and killed responsible for the jogger’s death? Removed bloody fur from lion’s lips, front paws for testing Autopsied lion, stomach contents included human scalp with hair still attached To Catch A Cougar 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 GEL: 9 Paw 10 Blank 11 Scalp 12 Claw 13 Victim 14 Ref. BLOT: Human DNA probe PCR Amplified DNA Type 4, 4 Array Technologies Method Substrate Detection Radioactive, Macroarray Nitrocellulose chemiluminescent, chromogenic Glass, nitrocellulose Microarray Fluorescent on glass High-density oligonucleotide Glass Fluorescent arrays Microelectronic Electrode grid Fluorescent arrays Microarray Technologies Method Array Application Comparative Detection of genomic Microarray, genomic hybridization amplifications and macroarray (CGH) deletions Detection of relative Microarray, Expression array changes in gene macroarray expression SNP detection, Detection of single- High density mutation analysis, base differences in oligonucleotide array sequencing DNA High-density Oligonucleotide Arrays Interrogate thousands of genes simultaneously. Requires a new array for each sample. Unlabeled probes are synthesized on the substrate. C A T A T A G C T G T T C C G (10–25mers) High-density Oligonucleotide Arrays Test DNA is fragmented before hybridization. Short fragments will bind specifically to complementary sequences on the array. Tiling (overlapping probe sequences) is used to blanket detection of nucleotide changes in the sample. High-density Oligonucleotide Arrays Fluorescent signal indicates which sample hybridized DNA to probe. Fluorescence is detected, normalized, and averaged by array readers and software. High-density Oligonucleotide Arrays Results displayed in graphical form. Normal sequence (TCG) A C G T del A C G T del A C G T del Heterozygous (TCG>TAG) A C G T del A C G T del A C G T del Represents five probes, each carrying the indicated base or deletion at the same position. Sequence-Specific Primer PCR (SSP-PCR) PCR primer extension requires that the 3′ base of the primer is complementary to the template. Primer G (Amplification) Normal template C G (No amplification) Mutant template T Sequence-Specific Primer PCR (SSP-PCR) Primer design is used to detect mutations in DNA. Generation of PCR product indicates the presence of mutation or polymorphism in the template. Detection of BRCA1 185delAG by SSP-PCR (1) GAAGTTGCATTTTATAAACCTT-> AAAATGAAGTTGTCATTTTATAAACCTTTTAAAAAGATATATATATA TGTTTTTTCTAATGTGTTAAAGTTCATTGGAACAGAAAGAAATGGAT TTATGTGCTGTTCGCGTTGAAGAAGTACAAAAT (2) ATTAATGCTATGCAGAAAATGTTAGAG-> (only in normal) GTCATTAATGCTATGCAGAAAATGTTAG[AG]TGTCCCATCTGGTAA (only in mutant)<--ATC - - ACAGGGTAGACCATT GTGAGCACAAGAGTGTATTAATTTGGGATTCCTATGATTATCTCCTA CAGT (3) TGCAAATGAACAGAATTGACCTTACATACTA <- CTTGTCTT AACTGGAATGTAT (4) Detection of BRCA1 185delAG by SSP-PCR +mm+ Product 230 bp (1) and (4) of primers (1) and (3) 180 bp (1) and (3) is specific for 120 bp (2) and (4) Mutation Allelic Discrimination Uses fluorescently labeled probes. Similar to Taqman technology. Generates “color” signal for mutant or normal sequence. Performed on real-time PCR instruments. Allelic Discrimination Probe complementary to normal sequence labeled with FAM fluorescent dye Probe complementary to normal sequence labeled with VIC fluorescent dye Normal Probe (FAM) Mutant Probe (VIC) Normal Green signal Mutant Red signal Allelic Discrimination Signals are detected and analyzed by the instrument software. Multiple samples are analyzed simultaneously. Het Mutant allele Mut (VIC) NL Normal allele (FAM) Restriction Fragment Length Polymorphism (RFLP) Restriction enzyme site recognition detects presence of sequence changes. e.g., G->A change creates EcoR1 site: NL: … GTCA GGGTCC GTGC… Mut: … GTCA GGATCC CTGC… NL Mut Het U C U C U C Agarose gel: U – uncut C – cut Heteroduplex Analysis with Single- Strand Specific Nucleases Uses nucleases that cut single–stranded bubbles in heteroduplexes. Region of interest is amplified by PCR. PCR product is denatured and renatured with or without added normal PCR product. Renatured duplexes are digested with nuclease; e.g., S1 nuclease Products are observed by gel electrophoresis. Heteroduplex Analysis with Single-Strand Specific Nucleases Mutation Mix, denature Renature Homoduplexes Heteroduplexes Heteroduplex Analysis with Single- Strand Specific Nucleases Homo duplexes Heteroduplexes not cleaved by enzyme cleaved by enzyme M NL Mutants Cleaved fragments indicate presence of mutation Invader Technology Mutation detection with proprietary Cleavase® enzyme. Sample is mixed with probes and enzyme. Enzyme cleavage of probe-test sample hybrid will yield fluorescent signal. Signal will only occur if probe and test sample sequence are complementary. Invader Technology Probes and enzyme are provided. 96-well plate format mut probe A wt probe G T Cleavage T A Complex formation (No cleavage) F Q A Cleavage F Detection Summary Mutations are heritable changes in DNA. Mutations include changes in chromosome number, structure, and gene mutations. Chromosomes are analyzed by Giemsa staining and karyotyping. Karyotyping detects changes in chromosome number and large structural changes. Structural changes include translocation, duplication, and deletion of chromosomal regions. More subtle chromosomal changes can be detected by metaphase or interphase FISH. Summary Mutations and polymorphisms are changes in the DNA sequence. DNA sequence changes have varying effects on the phenotype. Molecular detection of mutations include hybridization-, sequence-, or cleavage- based methods.