Download BIOL 212 General Genetics

BIOL 212 General Genetics
Spring 2007
Lecture 23: Recombinant DNA Revisited
Reading: Chap. 10 pp. 358-368
Lecture Outline:
1. Genetic Engineering Tools
2. Vectors
3. cDNA
4. Screening
Lecture:
1. Genetic Engineering Tools
Genetic Engineering, (aka cloning or recombinant DNA): fusion of DNAs from two
(or more) organisms.
Goals of G.E.:
 Purify rare gene
OR
 Prepare rare protein
G.E. relevance to humans:
 Map human genome
 Isolate disease genes
o Find out the proteins they encode
 Prepare medicines:
o Gene therapy: DNA
o Drug/hormone therapies: proteins
 Food, food products or additives: GMO organisms, peptides, proteins
Key tools of genetic engineering:
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Host organism: Especially bacteria, yeast
Vector: plasmid, virus, or artificial chromosome that carries the foreign DNA
Foreign DNA: Any organism
Restriction enzymes: “Cut DNAs”; site specific DNA endonucleases
DNA ligase enzyme: “Paste DNAs”; seal sugar-phosphate backbone of DNA
2. Vectors: carry foreign DNA, allow foreign DNA to be replicated and often also
transcribed and translated in recipient cells.
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Types of vectors:
 Plasmids
 Viruses
 Large capacity vectors: Cosmids, BACs, YACs
Plasmid vectors: Mostly derived from E. coli plasmids
Useful features of plasmid vectors
Ori site (plasmid origin of replication)
 Allows plasmid to replicate in bacterial host, separate from bacterial chromosome
 Derived from COLE1 plasmid of E. coli
Ampicillin resistance gene
Multiple cloning site (MCS), also called a polylinker
 Cleavage sites for many different restriction enzymes
Useful genetic marker such as lac Z gene or another antibiotic resistance gene
 Enables rapid selection of cells containing recombinant DNA vs. cells with vector
alone.
Fig. 10.9 pBluescript plasmid (commercial vector)
Steps used in cloning:
1. Cut foreign DNA with restriction enzyme
2. Cut vector DNA with same enzyme or one that produces compatible ends
3. Mix together, add DNA ligase to create recombinant DNA
4. Use transformation to introduce gene into bacteria
5. Genetic selection to distinguish recombinants from non-recombinants
6. Identify desired recombinant DNA (screening see below)
pBluescript: uses Blue/White color screen
Recombinant DNA: Foreign DNA is inserted into the lac Z+ gene  becomes lac ZScreen for beta-galactosidase by adding X-gal to the growth medium. Blue color
indicates X-gal is present. (Fig. 10.10)
Recombinant DNA present: colony is white
Non-recombinant plasmid vector: colony is blue
Virus vectors:
Most common are modified E. coli viruses
Example:
Lambda: replace middle region genes (lysogenic region) with foreign DNA
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Large capacity cloning vectors:
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Needed for cloning large genes of higher organisms (for DNA regions 50 kb or
larger) and for genome projects.
Can be modified forms of E. coli plasmids (cosmids), viruses (PACs), or F factors
(BACs) or can be artificial yeast chromosomes (YACs)
3. cDNA: Complementary DNA, a DNA copy of an mRNA
Most genes from higher eukaryotes are very large.
Length of mRNA much smaller than gene.
Much easier to study a DNA copy of an mRNA (called a complementary DNA)
Preparation of a cDNA: Fig. 10.8
a. purify mRNA by affinity chromatography (most eukaryotic mRNAs have poly A tail)
b. use reverse transcriptase, primer, and dNTPs to synthesize a strand of cDNA
c. remove the mRNA (treat with alkali or RNase)
d. use DNA polymerase I to synthesize the second strand of cDNA
OR use Taq polymerase, primers and PCR to make many copies of the cDNA by PCR
(this is RT-PCR or reverse transcriptase PCR)
cDNA can be cloned and sequenced (may be called EST, for expressed sequence tag)
4. Screening: Identify the recombinant DNA of interest
 Especially important when you start with many possible recombinants:
 A library consists of many bacteria, each with a different foreign DNA.
Genomic library: All the DNA of an organism
cDNA library: Copies of all the mRNA from a tissue or cell type
Hybridization is important technique used to isolate a particular DNA of interest
some examples:
 Use cDNA probe to mouse globin to detect the mouse gene
 Use a mouse gene probe to detect the human gene
Rapid screening, indirect:
Use PCR test or restriction digests to produce band of the size expected if the correct
DNA was cloned.
Final confirmation of recombinant DNA:
Use DNA sequencing and bioinformatics software.
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