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Lecture 22- DNA sequencing technologies, computer technologies, and a variety of other technologies have enabled the analysis of entire genome where we once were confined to studying one gene at a time.- Structural genomicso Organization and sequence of genetic information contained within a genome- Genetic mapso Proved a rough approximation of the locations of genes relative to the locations ofother know geneso Based on the recombinationo If recombination frequency is 50% -- loci are located on different chromosomes or far apart on the same chromosomeo <50% -- loci close together on same chromosomeo linked genes – rate of recombination is proportional to the physical distance between the locio distances on maps are measured in percent recombination, or map units- previously genes could be detected only by observing their influence on a traito genetic maps limited to single-locus traits by evidence of recombination- Limitations to genetic mapso Resolutiono Only approximations of real physical distances along the chromosome They are based on rates of crossing over, which vary from one part of chromosome to another- Physical mapso Based on the direct analysis of DNA, and they place genes in relation to distances measured in number of base pairso Connects isolated pieces of genomic DNA that have been clonedo Higher resolution and more accurate than genetic mapso Restriction mapping – determines the position of restriction sites on DNA DNA cut with restriction enzyme and fragments are separated by gel electrophoresis, the number of restriction sites in the DNA and distances between them can be determined by the number and positions of bands on the gel Doesn’t tell order or precise location of restriction sites To map—a DNA is cut with one r.e and another is cut with different r.e, third cut with both- Separted by gel electrophoresis and their sizes are compared- Overlap can be used to position restriction site on original DNA- Sequencing entire genomeo Size is the main issueo Usually can only measure small fragments – 500-700 bp—sequences at one timeo Difficulty in putting the short sequenced pieces of DNA back togethero Human Genome Project – map entire human genome Map-based strategy- Map-based sequencingo Short sequenced fragments are assembled into a whole-genome sequence by first creating detailed genetic and physical maps of the genome to provide locations of genetic markers (restriction sites, other genes, or known DNA sequences) at regularly spaced intervals along each chromosome Later makers used to align short sequenced fragments into their correct ordero After genetic and physical maps created – chromosomes are separated by pulsed-field gel electrophoresis  Standard g.e. cannot separate pieces this largeo Each chromosomes then cut up by partial digestion with r.e. Partial digestion – r.e. only allowed to act for limited time so not all sites in every DNA molecule are cut Produced a set of large overlapping DNA fragments which are then clonedo Large-insert clones put together in correct order on chromosome Method 1 relied on presence of high-density map of genetic markers A set of two or more overlapping DNA fragments that form a contiguous stretch of DNA = contigo Very difficult and slow processo Sequence fragments that allow you to infer the linear sequence of chromosomeo Also called Clone by Clone Sequencing- Whole-genome shotgun sequencingo Small-insert clones are prepared directly from genomic DNA and sequencedo Powerful computer programs then assemble the entire genome by examining overlap among the small-insert clones o These clones can be placed into plasmids which are simple and easy to manipulateo Fragment genome into workable sizes  clone fragments  sequence all cloneso Large amont of repeat DNA make assembly hardo Computer assembly is essential  this method much faster- Single-Nucleotide Polymorphismso Individual members of a species differ in a single base pair called this (SNP)o Arising through mutation, SNP inherited as allelic variants but do not cause phenotypic differenceso Arose from a single mutation on particular chromosome and then spread throughout population Differ at every 1000 nucleotides from each othero Each SNP is initially associated with other SNPs present on the particular chromosome on which mutation aroseo Specific set of SNPs on chromosome or part of chromosome called haplotype SNPs within haplotype are physically linked and tend to be inherited together Haplotypes can arise through mutation or crossing over, bring up particular set of SNPS in haplotypeo Nonrandom association between genetic variants within haplotype is called linkage disequilibriumo Use As markers in linkage studies (when close to disease-causing locus usuallyinherited together)  can reveal presence of genes that affect the diseaseo Genome Wide Association Studies (GWAS) – using SNPs to find genes Look at SNPs present – look for correlations with disease or other phenotypes. Close association might indicate the SNP is in or near a gene that plays a big role in determining that disease.o Compare SNPs between two groups – look for correlations (control group vs unknown)- Copy-number Variants (CNVs)o Differences among people in the number of copied of large DNA sequenceso Deletions, duplicationo Most contain multiple genes and potentially affect the phenotype by altering gene dosage and changing position of sequences which may affect regulation of nearbygenes- Expressed-Sequence Tag (EST)o If only protein-encoding genes of interest  mRNA examination instead of entireDNA genomic sequenceo RNA examined using ESTs—markers accociated with DNA sequences that are expressed as RNAo Isolate RNA from cell and using reverse transcription producing cDNA fragmentsthat correspond to RNA  Short stretched from ends of cDNA are sequences and called a tag which procides a maker that identifies the DNa fragment- Annotationo After a gene has been identified it must be annotated – linking its sequence information to other information about its function and expression – the protein tha it encodes, and information on similar genes in other specieso Start by applying genetic spelling and grammer rules Like find known consensus equences such as TATA boes, splice sites, open reading frameso Functional annotation – example use all mRNA to help identify expressed genes Indentification of all RNA

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UMD BSCI 222 - Lecture 22

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