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1 Lectures 26 and 27 recombinant DNA technology I. oal of genetics A. historically - easy to isolate total DNA - difficult to isolate individual gene B. recombinant DNA technology C. why get the gene? 1. determine nucleotide sequence 2. comparison of gene sequences 3. comparison with proteins of known function 4. can manipulategene and reintroduce 5. introduce gene into different species II. How to clone a gene using nucleic acid or antibody: A. isolate DNA 1. genomic DNA - break cells open under conditions that stabilize DNA - can use whole organism, or specific tissue - extract the DNA isolate DNA 2. cdna - isolate mrna, reverse transcribe to generate cdna isolate mrna B. break into fragments 1. why? chromosomes too large to work with 2. how? a. by restriction endonucleases prep cdna AATTC AATTC CT TAA C T TAA ATCC ATCC CCTA CCTA AATTC AATTC CTTAA CTTAA ATCC ATCC restriction endonuclease cleavage CCTA CCTA genomic DNA cleaved products 1

2 C. clone into vector 1. why? 2. we ll use a plasmid called puc as our example ampr - polylinker segment with DNA sequences recognized by different endonucleases vector 3. ligate DNA pieces into vector of choice a. purify plasmid b. cut vector appropriately c. ligate fragments into vector restriction endonuclease CTTAA AATTC + d. transform or transfect cells ligate D. get DNA clones into cells/bacteria by transformation 1. competent cells increase efficiency of transformation 2. some cells take up plasmid + 3. E. identify clone of interest what probe? 1. nucleic acid probe a. homologous gene from related organism b. purify protein, sequence protein, synthesize degenerate primers Phe Met Asn Asp ly Trp TTC/T AT AAC/t AC/t A/C//T T 2. antibody probe a. need to generate antibody b. use antibody to screen library 2

3 3. use of nucleic acid probe colony hybridization nucleic acid probe hybridizes via base pairing 1. colonies on plate 2. transfer to filter nucleic acid probe 3. lyse cells, denature DNA, incubate with 4.autoradiograph radioactive probe 5. identify positive colonies 4. use of antibody probe recognizes protein via high affinity binding 6. identify positive colonies 1. colonies on plate 2. transfer to filter antibody probe 3. lyse cells, incubate with antibody 4. incubate with secondary antibody 5.autoradiograph 6. identify positive colonies III. functional complementation A. library from type B. transform mutant C. select for type (i.e. transformant that has gotten vector + -type gene) D. this usually represents homologue of gene in question IV. positional cloning A. need to map gene to precise position on chromosome, and then B. few variations on basic approach 1. chromosome walk gnx yfg clone 1 clone 2 clone 3 3 clone 4 clones have the human gene

4 2. find clones that correspond to region of interest gnx yfg clone 1 clone 2 clone 3 C. Comparison of different methods. To use: 1. antibody probe clone 4 2. degenerate primers 3. nucleic acid probe 4. positional cloning 5. functional complementation V. Use clone A. look at gene organization by Southern blot 1. why? 2. a little background - agarose gel electrophoresis - charged molecules migrate in electric field - agarose results in separation by size 1300 bp 820 bp 1800 bp λ HindIII bp 500 bp 400 bp

5 3. back to Southern hybridization B. mrna production by Northern hybridization 1. yeast type probe yeastmutant yeast type yeastmutant what is it good for? 564 C. protein production by SDS-PAE and Western analysis (sodium dodecyl sulfate polyacrylamide gel electrophoresis) markers type mutant type overexpressor 1. separates protein by size 2. SDS used to denature protein and to eliminate affects of charge 5

6 markers type mutant markers type mutant 3. protein detection by Western blot a. protein in SDS-PAE can be transferred to membrane b. individual protein on membrane can be detected by specific antibody D. amplifying rare DNAs by polymerase chain reaction (PCR) 1. useful for: 2. how it works: 5' 3' 3' 5' 5' 3' 3' 5' E. DNA sequence determination 1. Maxam-ilbert 2. Sanger, based on chain termination a. uses dideoxynucleotide, not extended by polymerase 5 -ATCATTACCTAATTCATTAC-3 CATACCAT-5 A T C 5 -ATCATTACCTAATTCATTAC-3 CATACCAT-5 5 -ATCATTACCTAATTCATTAC-3 CATACCAT-5 5 -ATCATTACCTAATTCATTAC-3 CATACCAT-5 6

7 3. automated DNA sequencing A T C ATC 4. next generation DNA sequencing - uses variation of dideoxy method - massively parallel millions of reads simultaneously - in 2003, human genome sequence published - in 2011, human genome sequence 5. Ion semiconductor sequencing - monitors H+ given off during addition of nucleotide to growing DNA molecule - massively parallel millions of reads simultaneously - few days and ~ $2,000 VI. If we can sequence whole genome, can we identify genes by DNA sequence of mutant DNA? A. Strategy: sequence DNA of individual with genetic disorder, compare DNA sequence to type and look for mutation B. challenge: we all have polymorphisms (DNA sequence differences) C. How do we know which mutation causes the disease? 7

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