Lecture 2- Genome Analysis Tools (Chapt 2).pptx

Lecture 2- Genome Analysis Tools (Chapt 2).pptx - Lecture 2...

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Lecture 2 Tools for studying DNA Restriction enzymes blots PCR
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TECHNIQUES & TOOLS FOR STUDYING DNA Genomes are very large… - so need methods to obtain small (relatively speaking) sections of DNA in abundant & pure form for molecular analysis 1. Restriction enzyme cleavage & agarose gel electrophoresis 2. Southern hybridization & northern hybridization 3. PCR (polymerase chain reaction) & RT-PCR 4. Molecular cloning
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1. Restriction endonucleases “blunt” ends “sticky” ends Cohesive ends Fi g. 2. 1 0 DNA endonucleases cut double-stranded DNA at specific recognition sites - cleave DNA into specific, small fragments Bam HI: staggered cut with 5’ overhang
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4 types (I-IV) restriction endonucleases occur naturally Most commonly used are Type II Type II recognize a palindromic DNA sequence of 4-8 bp cut within the recognition site homodimeric require Mg2+ as cofactor Nomenclature : Example EcoRI E genus of producing strain co species of producing strain R additional strain descriptor I order of identification from strain EcoRI bound to major groove of DNA 5’-GAATTC-3’ 3’-CTTAAG-5’ Recognition sequences often 4 or 6 bp, but also “rare cutters” (eg. NotI 5’ GCGGCCGC 3’ ) can be useful for generating very large fragments in genomic mapping
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-“compatible ends” useful for cloning (eg. partial Sau3A genomic digest ligated into BamHI site in vector) Isoschizomers – restriction enzymes with identical recognition sequences … but may have different response to methylation state MspI cleaves 5’ CCGG 3’ regardless of methylation state HpaII does not cleave 5’ CCGG 3’ if 2d C is methylated Two different restriction enzymes may generate same “sticky ends” Useful for epigenetic studies (evaluating DNA methylation status near gene of interest)
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“Genome-wide DNA methylation analysis reveals novel targets for drug development in mantle cell lymphoma” Example using isoschizomers to assess DNA methylation state of genes in cancer patients Leshchenko et al. Blood 116:1025, 2010 - used assay called “ Hpa II tiny fragment Enrichment by Ligation–mediated PCR” - found “significant aberrancy in promoter methylation patterns compared with normal NBCs” log(HpaII/MspI) ratios NBC: naïve B cells (ie. from healthy people) Colour-coded figure: “heat map”
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Agarose gel electrophoresis Fig.T2.2 - to separate DNA fragments by size Fig.T2.1 Small fragments migrate more rapidly than large ones Lanes with size markers, on gels of different % agarose “Pulsed field” electrophoresis for separation of large DNA molecules (Fig.3.30) For example : restriction fragments generated by “rare cutters” - megaplasmids - whole chromosomes (eg. yeast)
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Lane 1 = uncut DNA Lane 2 = 6 bp cutter Lane 3 = 4 bp cutter so “continuum” of signals in lane 1 2 3 Why are different profiles expected for genomic DNA cleaved with Bam HI (6 bp cutter) vs. Sau 3A (4 bp cutter)?
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