Hum_Mol_Genet_Lecture_3_January_31_2012

Hum_Mol_Genet_Lecture_3_January_31_2012 - Mapping the Human...

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Mapping the Human Genome Mapping the Human Genome Genetic Mapping Physical Mapping DNA Sequencing
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Physical Mapping Systems Physical Mapping Systems Yeast Artificial Chromosomes (YACs) 200-2000 kb Bacteriophage P1 90 kb Cosmids 40 kb Bacteriophage λ 9-23 kb
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Large Fragment Cloning Large Fragment Cloning Insert Size --- Hybrid cells : multiple chromosomes : 100 -3000 Mbp --- Mono-chromosomal hybrids : 50- 300Mbp --- Sub-chromosomal hybrids : 1-50 Mbp --- Double-minutes : 0.3-2 Mbp --- Yeast Artificial Chromosomes : YACs : 0.05-2 Mbp --- BACs (F-plasmid derived) : - 300 kb --- PACs (large P1 clones) : - 300 kb --- T4-packaging system : - 120 kb ( - 300 kb) --- EBV derived vectors : - 180 kb --- P1 + P1 packaging : - 80- 90 kb --- Cosmid vectors : 35 - 45 kb --- Lambda replacement vectors : 8 - 22 kb --- Micro-dissection clones : - 1.5 kb
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Insertion Vectors cDNA cloning and expression GT10, GT11, Zap Replacement Vectors Genomic cloning EMBL3, EMBL 4 Bacteriophage Lambda Bacteriophage Lambda
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Insertion vector Small inserts (cDNAs) Expression cloning Lambda gt11 Lambda gt11
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Insertion vector Small inserts (cDNAs) Expression cloning In vivo excision of inserts Lambda Zap Lambda Zap
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Replacement vector Larger inserts Genomic DNA Spi selection Lambda Fix Lambda Fix
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Replacement vector Larger inserts Genomic DNA Spi selection Lambda EMBL3 Lambda EMBL3
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SORTED CHROMOSOME ISOLATION PROCEDURE SORTED CHROMOSOME ISOLATION PROCEDURE (1) Sort tubes are spun at 3 K rpm for 2 hours at 4 degrees Celsius. (2) The resulting pellet is extracted with Proteinase K/SDS; the sort tube is also extracted to isolate chromosomes adhering to the side of the tube. (3) Both samples are Phenol/CHCl 3 extracted and dialyzed overnight. SORT TUBE spun @ 3K rpm Proteinase K/SDS SUPERNATANT Proteinase K/SDS PELLET Proteinase K/SDS SORT TUBE Phenol / CHCl 3 Extract Phenol / CHCl 3 Phenol / CHCl 3 Use for purity 1% analysis of DNA and quantitation Clone into ==> 75-85% Lawrist of DNA Vector Clone into ==> 14-24% Charon of DNA Vector
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CONSTRUCTION OF LARGE INSERT LAMBDA LIBRARIES CONSTRUCTION OF LARGE INSERT LAMBDA LIBRARIES Package Amplify on E. Coli host strain K802 (rec A-) Charon 40 Vector (9 - 23 kb capacity) Polystuffer L arm R arm BamHI BamHI cos cos Polystuffer L arm R arm BamHI BamHI cos cos BamHI (1) Nae 1 (2) PEG precipitation (3) Phenol, chloroform extraction (4) Ethanol precipitation (5) BamHI Ligate Ba Ba Characterize Before Characterize Before Distribution to (ATCC) Distribution to (ATCC) Plate the Lambda library and screen with probes to identify phage which contain sequences of interest SOURCE DNA FOR CLONING SOURCE DNA FOR CLONING Grow cell (e.g. hybrid cells: J640-51) Grow cell (e.g. hybrid cells: J640-51) Isolate chromosomes by flow-sorting Isolate chromosomes by flow-sorting Purify chromosomes using described protocol
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This note was uploaded on 01/06/2012 for the course BIOL 4753 taught by Professor Batzer,m during the Fall '08 term at LSU.

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Hum_Mol_Genet_Lecture_3_January_31_2012 - Mapping the Human...

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