Recombinant DNA Technology

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Recombinant DNA Technology
 Various techniques have provided the means to analyze, modify, and
recombine various DNA sequences
 Much of this technology has been applied to agriculture
Tools of the trade
 Bacteria are nice enough to produce restriction endonucleases
 This makes it easy to construct recombinant DNA molecules
o Any DNA cleaved with EcoRI will have the same cohesive (sticky) end
o DNA ligase can then finish the job
What makes a good cloning vector?
 Plasmid vectors
Cloning genes in bacteria
 The purpose is to produce recombinant molecules containing genes of
interest
 We make use of restriction enzymes and ligase
 Delivery of recombinant DNA
o After the formation of the recombinant DNA molecules, they have to
be introduced into the bacteria
o Transformation
o The process of transformation in the lab is similar to transformation
in nature
o However, we usually have to use tricks to get the bacteria to take up
the DNA
o Electroporation and biolistics are other methods
 We can use other tricks to find the recombinant clones

Bacteriophage vectors
o High efficiency of delivery
o Can handle larger DNA inserts than plasmids
 We can also tweak the plasmids to ensure expression of the new gene
Cloning genes in eukaryotes
 Yeast artificial chromosomes (YACs) contain a yeast origin of replication, a
pair of telomeres, and a centromere
o These can carry inserted DNA fragments of 600-1000 kb
But how do we find that one gene?
 Partial restriction enzyme digestion is used to produce the genomic library
 The goal is that every segment of the genome will be represented several
times in the library
 Genomic libraries are created with the intent of having every portion of the
genome represented several-fold among many different clones

 cDNA libraries are made by first isolating the mRNA and using reverse
transcriptase to make cDNA copies
 Then the cDNA copies are cloned into a library

Now that we’ve made the library, we need to find the clones that contain the
gene
o We can use probes complementary to the desired gene sequence
o These are often homologous sequences from different organisms
Polymerase Chain Reaction
 We can amplify small target stretches of DNA by using the polymerase chain
reaction (PCR)
 This requires primers, Taq polymerase, and a thermocycler
After cutting DNA, how do we separate the resulting fragments?
Gel electrophoresis
 But cutting complex DNA with restriction enzymes generates a huge number
of DNA fragments
How can you locate specific fragments containing specific genes?
 Southern blotting with probes
 Probes are DNA or RNA molecules that are complementary to the sequence
of the gene of interest
 We can use gel electrophoresis to separate the DNA fragments based on their
size
 Southern blotting then allows us to transfer the DNA from the gel to a
membrane
 The double-stranded DNA can then be denatured and hybridized with the
probe
 Since there is Southern blotting, there is also northern blotting, western
blotting, etc.
DNA sequencing
 We can make use of our knowledge of DNA replication and a cool twist to
determine the sequence of DNA segments
 New techniques are being incorporated to increase the throughput of DNA
sequencing
DNA profiling
 Most DNA profiling makes use of microsatellites or short tandem repeats
(STRs) - short sequences tandemly repeated at various sites in the genome
 There is great variance in the number of copies at each site
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