Chapter 20: Gene isolation and manipulation

Recombinant (chimeric) DNA: fused DNA from two different organisms

Recombinant clone: vector (bacterial plasmid, virus) + insert (DNA fragment to be cloned) Recombinant (transgenic) organisms: host genome + DNA from another organism

(Arber & Smith: Nobel Prize,1978)

pallindromic sequences

Using restriction sites to create a recombinant molecule

GAATTC CTTAAG

only complementary ends can ligate!

Plasmid vector contains antibiotic-resistance marker

On antibiotic medium, only cells receiving a complete plasmid form colony (other ligation products will not permit host cell growth)

Grow and purify DNA from single colony

Using antibiotic resistance and lacZ markers to select plasmid-bearing colonies

Insertion disrupts lacZ (cell cannot hydrolyze X-Gal)

Using antibiotic resistance and lacZ markers to select plasmid-bearing colonies

Insertion disrupts lacZ (cell cannot hydrolyze X-Gal)

Bacteriophage lambda (): engineered as vector for cloning large DNA fragments Central 1/3 of genome (~45 kb) contains lysogenic function genes Can substitute ~15 kb cloned DNA into genome and the virus is still capable of lytic infection
e.g., the Drosophila genome (~150,000 kb) can be contained in

a minimum of 10,000 recombinant lambda clones (can fit on one 15 cm Petri plate)

Creating a genomic library in bacteriophage lambda

Useful for inserts 10-20kb

Fig 20-6

Fig 20-7

Useful for inserts 100-300kb -must use transducing phage to get into cell!

cDNA: complementary DNA; DNA complementary to RNA

Usually made against mRNA cDNA is essentially an intron-less copy of a gene, minus 5 and 3 flanking regulatory regions of the gene Prepared using reverse transcriptase (an RNAdependent DNA polymerase enzyme of RNA viruses)

Creating cDNA (DNA complementary to mRNA)

Creates clonable DNA copy of specific mRNA or can make cDNA library (representing mRNA population)

Remember:

no promoter no introns CDS + UTRs only

Variety of gene transfer mechanisms used to get recombinant molecules into host cells

Identifying a desired clone/gene in a library:

Use a probe (previously cloned DNA, oligonucleotide, or antibody)

Detecting & isolating a specific clone within a library by hybridization

Using an antibody to detect & isolate a specific clone within a library

Fig 20-10

Agarose gel electrophoresis separates DNA fragments by size:

restrict cloned DNA electrophoresis stain with ethidium bromide visualize under UV Example: clones from a plasmid library digested with the restriction enzyme used to make the library

Southern/Northern blot analysis

agarose gel electrophoresis

transfer to nitrocellulose

hybridize with radioactive probe

autoradiograph to detect bands containing the probe sequence

Using restriction sites as markers to map a DNA fragment

(Nathans: Nobel Prize,1978)

Polymerase chain reaction (PCR)

Uses heat-stable DNA polymerase (e.g., Taq polymerase) Requires two opposite-strand primers; ~100 bp - ~3 kb apart on the target template Uses a regimen of temperature cycling to amplify the DNA target between the two primers

(Mullis: Nobel Prize,1993)

Polymerase chain reaction: Specific primers permit amplification of a DNA segment

* * * * * * * *
Subsequent cycles preferentially amplify primer-primer fragment

Dideoxynucleotide used for Sanger dideoxy DNA sequencing

(Berg, Gilbert & Sanger: Nobel Prize,1980)

Sanger dideoxy DNA sequencing

Fig 20-16

Automated sequencing readout of Sanger dideoxy DNA sequencing

Table 20-1

prospects for genetic engineering?

Variety of means of creating transgenics

Yeast: replacement and disruption alternatives

gene replacement

gene disruption

yeast are very recombinogenic readily integrate fragments into the genome by homologous exchange

Ti plasmid: a vehicle for making transgenic plants

wild-type Ti plasmid

recombinant plasmids containing T-DNA borders transferred into plant cells

Fig 20-26

T-DNA insertion is inherited as a Mendelian dominant marker

Transgenesis in Drosophila utilizes: utilizes a transposable element DNA constructs containing desired gene and P element components

Injection into embryos (P insertions into the genome)

Results in insertion of desired gene at random genomic sites

Engineering of mammalian genomes

Insert a gene (relatively easy) Disrupt a gene (knockout) Replace a gene (e.g., gene therapy)

Ectopic transformation of mouse embryos

First Step

Insertions at random (non-homologous) sites

mammalian cells are very recombinogenic readily integrate fragments into the genome (non-homologous & homologous exchange)

Making a targeted mutation (knockout) in mouse cells

First Step

Fig 20-31

Making a targeted mutation (knockout) in mouse cells

First Step

Fig 20-31

Using embryonic stem cells to make a knockout mouse

Second Step

Third Step

Cappechi, Evans & Smithies, Nobel Prize in Physiology or Medicine, 2008

Fig 20-32

Engineering of mammalian genomes

Insert a gene (relatively easy) Disrupt a gene (knockout) Replace a gene (e.g., gene therapy)

Gene insertion therapy of lit mice

autosomal dominant

Fig 20-33

Complications arising with germline gene therapy to cure genetic diseases in mammals is that most transgene integration events are random (not targeted)
Transgene does not replace defective gene (just complementation/transgene rescue) Transgene insert might disrupt another gene (creating an undesired mutation) Transgene usually segregates independently from the disease-causing gene

Alternatives in gene therapy

e.g., transgene on viral vector

Fig 20-34