Lectures 26 and 27– recombinant DNA technology
I. Goal 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
2. cDNA
- isolate mRNA, reverse transcribe to generate cDNA
B. break into fragments
1. why? chromosomes too large to work with
2. how?
a. by restriction endonucleases
1
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
3. ligate DNA pieces into vector of choice
a. purify plasmid
b. cut vector appropriately
c. ligate fragments into vector
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
2
a. homologous gene from related organism
b. purify protein, sequence protein, synthesize degenerate primers
Phe
Met Asn Asp
Gly
Trp
TTC/T ATG AAC/t GAC/t GGA/C/G/T TGG
2. antibody probe
a. need to generate antibody
b. use antibody to screen library
3. use of nucleic acid probe – colony hybridization – nucleic acid probe “
hybridizes” via base pairing
4. use of antibody probe – recognizes protein via high affinity binding
III. functional complementation
A. library from wild type
B. transform mutant
C. select for wild type (i.e. transformant that has gotten vector + wild-type gene)
D. this usually represents homologue of gene in question
3
IV. positional cloning
A. need to map gene to precise position on chromosome, and then…
B. few variations on basic approach
1.
chromosome walk
2. find clones that correspond to region of interest
C. Comparison of different methods. To use:
1. antibody probe
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?
4
2. a little background
- agarose gel electrophoresis
- charged molecules migrate in electric field
- agarose results in separation by size
3. back to Southern hybridization
B. mRNA production by Northern hybridization
1.
5
2. what is it good for?
C. protein production by SDS-PAGE and Western analysis
(sodium dodecyl sulfate – polyacrylamide gel
electrophoresis)
1. separates protein by size
2. SDS used to denature protein and to eliminate
affects of charge
3. protein detection by “Western” blot
a. protein in SDS-PAGE 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:
6
E. DNA sequence determination
1. Maxam-Gilbert
2. Sanger, based on chain termination
a. uses dideoxynucleotide, not extended by polymerase
5’-ATGCGGATTACCGTAATGTCATGGTAC-3’
CAGTACCATG-5’
5’-ATGCGGATTACCGTAATGTCATGGTAC-3’
CAGTACCATG-5’
5’-ATGCGGATTACCGTAATGTCATGGTAC-3’
CAGTACCATG-5’
5’-ATGCGGATTACCGTAATGTCATGGTAC-3’
CAGTACCATG-5’
3. automated DNA sequencing
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
7
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 wild type and look for mutation
B. challenge: we all have polymorphisms (DNA sequence differences)
C. How do we know which mutation causes the disease?
8