Biology 212 General Genetics
Lecture 14 Recombinant DNA I
Spring 2007
Reading: Chap. 6 pp. 224-233
Lecture Outline:
1. Restriction enzymes
2. Gel electrophoresis and Southern blot
3. Polymerase chain reaction (PCR)
4. Dideoxysequencing
Lecture:
 Recombinant DNA encompasses a number of different methods for altering and
detecting DNA. A recombinant DNA molecule consists of a foreign DNA
inserted in a vector.
 Many of these methods depend on use of E. coli enzymes such as DNA
polymerase I as specific tools.
1. Restriction enzymes
 discovered in bacteria, blue-green algae
 site-specific, most recognize 4-6 bp sequence
 are endonucleases--cut within a DNA molecule
-----G GATC C-----------------------C CTAG G-------------------
Recognized by restriction enzyme BamHI
Cleavage produces 4 bp overhang
Uses of restriction enzymes
Fragment DNA of an organism into smaller pieces
Separate by electrophoresis
to study particular region
Insert into vector to create a recombinant DNA
2. Gel electrophoresis and Southern blot
Agarose gel electrophoresis
 Separate molecules in electric field
 Smaller molecules travel faster
 Larger molecules travel more slowly
 DNA is negatively charged and is attracted toward the positive pole
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Construct a physical map of DNA (restriction map) by:
 digesting DNA with restriction enzymes
 separating DNAs by gel electrophoresis
 determining sizes of fragments by comparison to known fragments
 physical mapping data mostly additive
Example:
Construct restriction map of linear DNA:
EcoRI
BamHI
4 kb
6 kb
2 kb
Southern blot: Transfer of DNA fragments from a gel to a membrane for
hybridization with a probe. Specific DNA fragments can be identified in a mixture.
Fig. 6.27
Hybridization:
 Based on base pairing of complementary DNA or RNA sequences
 Prepare hybridization "probe", a DNA or RNA sequence to gene of interest,
usually tagged with a radioactive nucleotide
 Incubate with Southern blot, wash off excess
 Hybridizing fragments detected by autoradiography
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3. Polymerase chain reaction (PCR)
 method for reproducing DNA
 means to obtain large quantities of a specific DNA sequence
 requires
o small amount of starting DNA
o forward and reverse primers
o deoxynucleotide triphosphates
o thermostable DNA polymerase
o appropriate buffer conditions
 involves multiple cycles (chain reaction)
Process of PCR
a. Denaturation: heat at 94˚C
b. annealing 45-65˚C
c. extension 72˚C
Special applications of PCR:
Forensics: reproduce DNA from as little as a single cell
DNA diagnostics: very little starting sample needed to perform a DNA test to diagnose a
genetic disease or identify carriers
4. Dideoxy DNA sequencing
method for determining order of bases on DNA
uses modified nucleotides as chain terminators in a DNA synthesis reaction
Fig. 6.29 compare structure of deoxy and dideoxynucleotide
Requirements of reaction:
 Primer
 Template
 Mixture of deoxynucleotides
 Dideoxynucleotide
 Synthesize chains of various lengths
 Terminate at dideoxynucleotide (for example, ddA)
 Carry out similar reactions for all four nucleotides
 Separate products by gel electrophoresis
 Smallest products travel farthest--sequence closest to primer is at bottom of gel
Advances in DNA sequencing technology
 Automated process
 Fluorescent tags to distinguish 4 different bases
 Scan gel during electrophoresis
 Produce colored trace
 Sequence data can be analyzed and assembled using high speed computers
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