* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 1 Mbp DNA for human genome
Transposable element wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
DNA sequencing wikipedia , lookup
DNA barcoding wikipedia , lookup
DNA polymerase wikipedia , lookup
Genome (book) wikipedia , lookup
Whole genome sequencing wikipedia , lookup
Primary transcript wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
DNA profiling wikipedia , lookup
Human genetic variation wikipedia , lookup
Genetic engineering wikipedia , lookup
Genome evolution wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
DNA vaccination wikipedia , lookup
Designer baby wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Point mutation wikipedia , lookup
Metagenomics wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Human genome wikipedia , lookup
Molecular cloning wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
DNA supercoil wikipedia , lookup
Epigenomics wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Genome editing wikipedia , lookup
Non-coding DNA wikipedia , lookup
SNP genotyping wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genomic library wikipedia , lookup
Microevolution wikipedia , lookup
History of genetic engineering wikipedia , lookup
MAPPING GENOMES – genetic, physical & cytological maps Genetic distance (in cM) 1 centimorgan = 1 map unit, corresponding to recombination frequency of 1% Physical distance (in bp) - determined by DNA sequencing (or restriction fragment sizing) 1 cM corresponds to ~ 1 Mbp DNA for human genome Gibson Fig. 1.3 Discrepancies (at times) between genetic & physical maps Genetic mapping data may have limited resolution (eg if not enough progeny) or limited accuracy (eg recombinational “hotspots”) or “coldspots” - no absolute relationship between genetic & physical distances Yeast chr III Fig. 3.25 Why don’t tomatoes taste as good as they used to? Sl-GLK2 gene mutation improves uniformity of red colour… but less sugar & less tasty Nature May 31, 2012 Ottawa Citizen September 15, 2012 Powell, Science 336:1711, 2012 STRATEGIES USED IN GENOMIC ANALYSIS - because of recent advances in rapid sequencing technologies & powerful bioinformatics tools, “shotgun” approach is now usually used - except for genomes with high amounts of repetitive DNA Markers used to anchor assembled sequences on map Fig. 3.3 DNA MARKERS USED IN GENETIC MAPPING RFLPs – restriction fragment length polymorphisms SSLPs – simple sequence length polymorphisms SNPs – single nucleotide polymorphisms DNA marker must have (at least) two different alleles to be useful in monitoring inheritance patterns Alleles – alternative forms of a gene (or DNA sequence) at a particular locus (chromosomal site) Polymorphisms – occurrence of two or more variants (alleles, phenotypes, sequence variants) at significant frequencies in a population if present < 2% in population, called “mutation” or “mutant allele” Haplotype – set of alleles linked on a chromosome usually inherited together as a block, “haploid genotype” 1. RFLPs (Restriction Fragment Length Polymorphisms) - inherited pattern of restriction sites follows Mendelian rules Fig. 3.4 How to detect RFLPs? i. by Southern hybridization analysis - if “polymorphic” restriction site is present, detect two hybridizing fragments, vs. one if site is absent Length of large fragment = sum of two small ones Fig. 3.5 ii. by PCR analysis Lane 1 = size markers Lane 2 = DNA homozygous for allele 1 Lane 3 = DNA homozygous for allele 2 Fig. 3.5 2. SSLPs (simple sequence length polymorphisms) Microsatellites - tandem repeats of short stretches ( < 5 bp or so) in clusters usually < 150 bp Different number of GA copies in tandem Fig. 3.6 How to detect SSLPs by PCR Interpretation if lane A = DNA from person #1 and lane B = DNA from person #2 ? Profile for lane A sample in blue & size markers in brown The more copies in tandem, the larger the PCR product ... and the slower it migrates electrophoretically Fig.3.5 Differences in copy number in microsatellite array among individuals useful in genetic profiling DNA fingerprinting, forensic analysis homozygous vs. heterozygous state for a particular microsatellite locus? Fig. 7.24 Blue, Green: fluorescent tagged PCR products Red: DNA size markers Each lane = DNA profile (with microsatellite length variants) from different individual - high heterozygosity of microsatellites (multiple alleles in population) - estimated that ~ 3 million differences between any two copies of human genome (natural sequence variation) 3. SNPs (single nucleotide polymorphisms) SNPs usually exist as just two variants in population, because for a third allele to arise, mutation must occur at exactly the same position (see p.69) Fig. 3.7 “...found population-specific allele frequency changes.... one SNP in EPAS1 (transcription factor gene involved in response to hypoxia) showed a 78% frequency difference between Tibetan [high altitude] and Han [sea level] samples.” Exome = “coding sequences of 92% of human genes” “Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.” Yi et al. Science 329:75 (July 2 & August 13, 2010) How to detect SNPs Oligomer-specific hybridization tests - reduced stability of hybrid between oligomer probe & test DNA if any mis-matches - “stringency” of hybridization conditions (eg.temperature, salt…) influences stability of imperfect complementarity Fig. 3.8A (i) Detecting hybridization by “dye-quenching” “molecular beacon” FRET (fluorescence resonance energy transfer) - when oligomer probe forms stable hybrid with template DNA (vs. hairpin structure), generates fluorescent signal NB: your text just shows a figure for this type of hybridization probe, but others include tagged PCR products (ss), end-labelled oligomers, etc. see Topic 3 Fig.3.8B (ii) Detecting oligomer hybridization using DNA microarrays - for large scale analysis of SNPs (see later Topics for other applications) Tech.note 3.1 “Oligo chips” - density ~ 10 6 oligos/cm2 - grid of different oligomers synthesized in situ on glass wafer by photolithographic process - fluorescently labelled DNA (or cDNA…) allowed to hybridize - hybrids monitored by laser scanning “~150,000 polymorphisms can be typed in a single experiment...” p.71 Affymetrix's GeneChip Applications for drug discovery: World Array 1: Axiom® Genome-Wide EUR 1 Array Plate – European ancestry World Array 2: Axiom® Genome-Wide EAS 1 Array Plate – East Asian ancestry etc. “SNPs selected from disease and drug response GWAS databases [from Genome-Wide Association Studies] … saturated coverage in over 5,000 gene regions previously identified as disease-associated…. ” Griffiths p.404 Why might some signals be stronger than others in SNP analysis? Note: microarray Fig.T3.2 (p.71) shows RNA expression data not SNP data Advantages of DNA markers - easy to generate large number & easy to detect Genetic markers - SNPs & microsatellites rather evenly spread in genome - codominant, can detect both alleles in heterozygote Microsatellite analysis by PCR Fig.7.24 Fig. 3.19 & 3.21 DNA markers PHYSICAL MAPPING APPROACHES 1. Restriction mapping - determine relative positions of restriction sites in DNA molecule by analyzing sizes of fragments generated by restriction enzymes How many EcoRI sites are present? Where? ... Fig. 3.27 2. FISH (fluorescence in situ hybridization) - intact metaphase chromosome (as ss DNA, denatured with formamide) hybridized with fluorescently labelled probe ~ 1 Mbp resolution of markers Ottawa Citizen newspaper Sept. 6, 2012 (see Topic 1) 46 human chromosomes telomere-specific repetitive DNA probe Fig. 3.32 Fibre-FISH - mechanically-stretched chromosomes or “molecular combing” of isolated DNA for higher resolution 10 – 25 kb resolution Fig. 3.31 Probes: cosmid 1, cosmid 2, (overlap region in yellow) Nature Feb. 2001 3. STS (sequence tagged site) physical mapping What are STSs? - any short, known DNA sequence (100-500 bp) which is unique in genome … so can be used as physical mapping marker A B C D E F G HI J K L MN O P Q R S T U V W STSs BACs - wish to have many STSs along chromosome - if STS is present in two clones, can deduce that those clones are overlapping Examples of sequences that can serve as STSs 1. ESTs – expressed sequence tags (from cDNA clones, ie. representing mRNAs for various genes see Fig.3.36) 2. SSLPs (Microsatellites) - tandem simple sequence repeats with unique flanking sequences 3. Random genomic sequences as long as single copy in genome eg. only one hybridization signal in Southern experiment Distance between 2 STSs will determine whether they are: likely, sometimes or never located on same DNA fragment In genomic analysis, want overlapping set of DNA fragments spanning genome (or chromosome separated by flow cytometry, Fig.3.38) (eg. clone library) Fig. 3.35 1. Clone libraries Fig. 3.39 Fig. 3.1 Potential problems in mapping genomes with repetitive DNA 1. Tandem repeats (DNA sequences shown as single stranded, eg. from computer file) - omission of internal regions of tandem repeat array… Fig.3.2A 2. Dispersed repeats - omission of sequences located between two repeats or incorrect linking (eg from different chromosomes) Fig. 3.2B