* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Introduction to Genetics and Genomics
Zinc finger nuclease wikipedia , lookup
DNA profiling wikipedia , lookup
Metagenomics wikipedia , lookup
Transposable element wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Minimal genome wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Public health genomics wikipedia , lookup
DNA vaccination wikipedia , lookup
DNA polymerase wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Oncogenomics wikipedia , lookup
Human genome wikipedia , lookup
Point mutation wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Primary transcript wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genetic engineering wikipedia , lookup
Epigenomics wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
SNP genotyping wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Genome (book) wikipedia , lookup
Molecular cloning wikipedia , lookup
DNA supercoil wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Genome evolution wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Genomic library wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Designer baby wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Microsatellite wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Genome editing wikipedia , lookup
Helitron (biology) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Introduction to Genetics and Genomics 51:123 Terry Braun 1 Outline • Basic Mendelian Genetics – Mendel’s laws • independent assortment • independent segregation – mitosis and meiosis – PCR and markers – dominant/recessive and pedigrees • genotype and phenotype – alleles • Basic molecular genetics – – – – DNA RNA proteins Central Dogma • genes and gene structure – cells and chromosomes Principles of Genetics, Tamarin, Human Molecular Genetics 2, Strachan and Read 2 KeyTerms • marker – a region of the genome that may often be uniquely identified and distinguished between individuals • minisatellite – a type of marker that varies in length from 14 to 100 nucleotides • microsatelite – a type of marker that is very short (2, 3, 4, 5, 6 nucleotides) -- aka STRP's (short tandem repeat polymorphisms) • polymorphism – a sequence variation • SNP -- single nucleotide polymorphism • polymerase chain reaction (PCR) – a reaction that mimics DNA duplication in meiosis (aka DNA amplification) (Kary Mullis) • DNA polymerase – a molecule that is essential for DNA duplication (and PCR) • primer – a piece of DNA that is essential for starting DNA replication (and PCR) • genotype – the genetic state of an individual (typically represented by a marker) 3 Genetic Marker – A genetic marker allows for the observation of the genetic state at a particular genomic location (locus). • A genotype is the measured state of a genetic marker. • A tool for observing inheritance patterns (Mendel's rules and meiosis) • May never be feasible to sequence cases directly, however the current cost is decreasing – An “informative” marker is often “heterogeneous, or “polymorphic” and enables the observation of the inheritance of genetic material. 4 Example -- genotypes Pedigree male female parents offspring 11 11 uninformative 11 11 12 14 34 24 heterogeneous These labels (markers) are a measure of the genetic state of each individual. Recall from "Rule of Segregation", offspring get one gene from each parent. Markers are not genes, but they are regions on chromosomes (meiosis). 5 What a marker looks like in the Genome Geneticists assign numerical values to different versions of markers 6 Sources of Markers in the Genome • duplications • unequal homologous recombination • slippage and errors during DNA duplication 7 Duplicating DNA – to Use Markers to "Probe" Genomes of Individuals • mitosis is process that copies DNA in biology • the first step is to "unzip" the 2 strands of the double helix (DNA) • an enzyme called DNA polymerase makes a copy by using each strand as a template • two other components – nucleotides (A, G, T, C) (A-T, G-C, etc) – a short stretch of DNA called a "primer" (to prime the process) 8 PCR – Polymerase Chain Reaction • PCR is a process that copies DNA exponentially • mimics the process by organisms, but in vitro (in a test tube) • relies on the ability of DNA-copying enzymes to remain stable at high temperatures • Necessary components (in a vial) – – – – piece of DNA to be copied large quantities of four nucleotides large quantities of primer sequence DNA polymerase (Taq – named for Thermus aquaticus, a bacterium that lives in hot springs) 9 PCR Reaction • The reaction can be carried out entirely in a vial simply by changing the temperature – separate the 2 strands (in DNA) • heat to 75-90 C (165 F) for 30 seconds • this "melts" the DNA apart – the base pairing comes undone – "anneal" the primers • primers cannot bind to the template strands at such high temp – cooled to 55 C for 20 seconds – make complete copy of template (and thus new templates for the next cycle) • Taq polymerase works best at 75 C (hot springs) • nucleotides are added (complement – if template has A, T is added, etc) 10 PCR Reaction • Three steps – separation of strands – annealing of primers to template – synthesis of new strands • • • • Takes approx. 2 minutes Each reaction is carried out in the same vial, and after every cycle, each piece of DNA is duplicated (exponential copying) Cycle can be repeated 30 times (2^30 = 1,073,741,824) 1 million copies can be made in approximately 3 hours from a single copy of DNA – this is why very minute samples can be used to identify individuals in crime scene investigations • • • Valuable tool to multiply unique regions of DNA so they can be detected in LARGE genomes Note, we need to know the flanking sequence to be able to design primers Also, this flanking sequence needs to be unique otherwise the reaction could amplify sequence from multiple regions of the genome 11 Exponential Nature of Reaction 12 Sequencing Reaction 13 Automated 14 Components of the Reaction 15 DNA polymerase (Taq) and Synthesis 16 17 Animations • http://allserv.rug.ac.be/~avierstr/principles/ pcrani.html 18 Markers – the early days • Prior to the HGP, markers were (and still are) valuable tools for observing inheritance patterns • Investigators consumed considerable time and resources identifying markers • Some markers were observed in a test group of individuals to asses quality, and heterogeneity. – CEPH (Centr d'Etude du Polymorphisme Humain) • Affymetrix SNP Chip -- 500,000 SNPs (~$450 -2007) 19 Marker GATA50G06/D15S643, Genotypes, and primers – 133101: 215, 197 133102: 219, 215 Genomic chr15 : ttctgctctt ccctactttg ATACCTGGAG ctcttatcct tgtctgtcta tctatctatc AGGTTTTAAA tctatctatc atctgtcacc ttgtctaaaa ccgttgctgc TCCTTGGTCC tggggacaga tctatctatc tatctatcta GCTGTTatcc tatctatcta tattta tgtcagtcta ctggctatac ttcttgggaa ttaaaccctt tgtctatcta cctacctaac ttggggacag tctatctatc aatccttact cttgtattta aaagtattga aaactatcta tctatctatc tacctaccaa attaaaccct tatctatcta tgtaattgtg ttgctggcct ggttttaaag tctgtctgtc tatctatcta aaaaGCATTG caaccctcta tctatctatc 57501064 57501114 57501164 57501214 57501264 57501314 57501364 57501414 http://genome.ucsc.edu/cgibin/hgc?hgsid=76756345&o=57501058&t=57501337&g=stsMap&i=GATA50G06& c=chr15&l=57401058&r=57601337&db=hg18&pix=800 http://research.marshfieldclinic.org/genetics/genotypingData_Statistics/genotypes_ 20 referenceIndividuals.asp Marker GATA50G06/D15S643, Genotypes, and primers – 133101: 215, 197 133102: 219, 215 Genomic chr15 : ttctgctctt ccctactttg ATACCTGGAG ctcttatcct tgtctgtcta tctatctatc AGGTTTTAAA tctatctatc atctgtcacc ttgtctaaaa ccgttgctgc TCCTTGGTCC tggggacaga tctatctatc tatctatcta GCTGTTatcc tatctatcta tattta tgtcagtcta ctggctatac ttcttgggaa ttaaaccctt tgtctatcta cctacctaac ttggggacag tctatctatc aatccttact cttgtattta aaagtattga aaactatcta tctatctatc tacctaccaa attaaaccct tatctatcta tgtaattgtg ttgctggcct ggttttaaag tctgtctgtc tatctatcta aaaaGCATTG caaccctcta tctatctatc 57501064 57501114 57501164 57501214 57501264 57501314 57501364 57501414 21 Genome to Gene Sequence Markers are typically NOT genes, however they may reside in the genome relatively close to a gene. 22 Basis for Inheritance of Disease: Examples Aa Pedigree Aa male female parents offspring Aa 1/2 A Aa AA 1/2 a 1/2 A 1/4 AA 1/4 Aa 1/2 a 1/4 Aa 1/4 aa P(AA) = 1/4 P(Aa) = 1/2 P(aa) = 1/4 AA Aa A from mom/dad? a from mom/dad? 23 1 2 3 4 5 6 232 234 236 238 240 242 1 2 3 4 5 Examples 234 236 238 240 242 234 238 232 238 14 24 3 234, 238 234, 238 238, 238 238, 232 234, 232 If you "genotype" an individual at enough markers, you can calculate the probability of uniquely identifying an individual. Note that the lawyers for OJ Simpson argued that "recoded" allele numbers increased the likelihood of contamination and false identification. 24 Examples Affected individuals 25 Examples Dominant model Geneticists then look for genes that mimic this pattern of inheritance 26 Example Recessive model. Very unlikely, because "founders" marrying in also carry the disease, which by definition is a rare genetic disorder. 27 BBS4 Pedigree 28 Monogenic and Polygenic Diseases – monogenic (Mendelian) -- one gene • “simple” (dominant and recessive) Mendelian inheritance • direct correspondence between one gene mutation and one disorder • majority of disease genes found are monogenic – polygenic -- (complex) multiple genes • heterogeneity – disease caused by multiple genes • epistasis – disease caused by multiple interacting genes • obviously finding these is harder -- but why??? 29 ...Mongenic and Polygenic Diseases • phenocopy • reduced penetrance – Example -- sickle cell anemia • • • • “classic” recessive disorder defect in red blood cells (hemoglobin) but… infant hemoglobin gene can “leak” wide range of phenotypes 30 Bardet-Biedl Syndrome (BBS) • Obesity – Diabetes/ hypertension • • • • • • Retinopathy Hypogenitalism Polydactyly Mental Retardation Renal Anomalies Heart defects Rare disorder, but common phenotypes 31 Molecular Analysis of BBS • • • • • • • • BBS1 - 11q13 BBS2 - 16q22 BBS3 - 3p13 BBS4 - 15q21 BBS5 - 2q31 BBS6 - 20p12 BBS7 - 4q27 BBS8 - 14q31 Novel* Novel* Novel†, TPR Repeats Type II Chaperonins Novel* Novel†, TPR Repeats *,† - Some Similarity 32 33 Some Useful Properties of DNA • fragments of DNA have a minute negative charge – if you apply an electric field to DNA in a matrix, it will migrate to the positive pole • DNA is a linear molecule, but it tends to fold up (similar to a knot) – this bound up molecule of DNA will have a unique crosssectional area profile that is dependent on its sequence • Gel electrophoresis – DNA is placed in a polyacrylamide gel and a voltage is applied – polyacrylamide gel and pool analogy – applied charge will cause DNA to migrate dependent on its size, and its sequence 34 BBS4 Deletion (by PCR) Example of Usage exons 3 4 35 Molecular Genetics • Not covered – molecular details of DNA duplication • continuous replication, discontinuous, Okazaki fragments, etc. 36 Genome – so now we know where it comes from biologically – at least most of it • mitochondria – organelle of eukaryotes – number varies per cell – 10 to 10K – human mitochondria is 16,569 nts – mostly coding (no introns???) – duplex strand and circular – inherited maternally only • consequences – mito thought to be originally free-living bacteria – origins (one or multiple events?) 37 Leber Optic Atrophy • LHON – mid-life, central vision loss – caused by missense mutations in mtDNA – generally familial 38 • Evolution of the mitochondrial genome and origin of eukaryotic cells 39 END 40 Another Marker? BRCA1-A good predictive marker of drug sensitivity in breast cancer treatment? * Mullan PB, * Gorski JJ, * Harkin DP. Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, BT9 7AB, United Kingdom. There are currently only two predictive markers of response to chemotherapy for breast cancer in routine clinical use, namely the Estrogen receptor-alpha and the HER2 receptor. The breast and ovarian cancer susceptibility gene BRCA1 is an important genetic factor in hereditary breast and ovarian cancer and there is increasing evidence of an important role for BRCA1 in the sporadic forms of both cancer types. Our group and numerous others have shown in both preclinical and clinical studies that BRCA1 is an important determinant of chemotherapy responses in breast cancer. In this review we will outline the current understanding of the role of BRCA1 as a determinant of response to DNA damaging and microtubule damaging chemotherapy. We will then discuss how the known functions of this multifaceted protein may provide mechanistic explanations for its role in chemotherapy responses. 41 Hardy-Weinberg Equilibrium • Rule that relates allelic and genotypic frequencies in a population of diploid, sexually reproducing individuals if that population has random mating, large size, no mutation or migration, and no selection • Assumptions – allelic frequencies will not change in a population from one generation to the next – genotypic frequencies are determined in a predictable way by allelic frequencies – the equilibrium is neutral -- if perturbed, it will reestablish within one generation of random mating at the new allelic frequency • Ideal case 42 Expected allele frequencies Deviations from distribution may indicate special cases. 43 H-W • f(AA) = p2 • f(Aa) = 2pq • f(aa) = q2 • (p+q)2 • (p2 + q2 + r2 + 2pq + 2pr + 2qr)= (p+q+r)2 44 Use of H-W • • • All other things being equal, we can "expect" that the distribution of genes in a subset of a population would be represented by the distribution of genes in the population Deviations from this expected distribution is evidence of selection or enrichment Association – when a specific variation of a gene (allele) is correlated with a phenotype (or disease, or trait) more frequently than you would expect by H-W – also called Linkage Disequilibrium (since genes are normally in equilibrium) Often used to evaluate validity of an assay. For example, let us say that I genotype 400 people at a marker with 2 alleles (A and B). I observe the following genotypes: marker1: AA: 36 AB 168 BB 196 marker2: AA 2 AB 37 BB 360 marker3: AA 64 AB 144 BB 192 Which maker is suspicious? 45 Will return to Linkage in Later Lectures 46 47 48