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A. Recombinant DNA technology's success is due to a variety of factors including...
1. Enzymes such as...
A. Restriction enzymes (restriction endonucleases) were discovered in the late 1960s by
Werner Arber, Hamilton Smith and Daniel Nathans.
B. Many prokaryotic restriction enzymes recognized specific bases sequences (four-eight
base pairs) in double-stranded DNA and cut the DNA.
C. The cleavage sites are at palindromic sequences and result in "sticky tails."
D. Common restriction enzymes include BamH1 and EcoR1.
E. Restriction enzymes can be used to "fingerprint" DNA molecules.
III. Electrophoretic Analysis of DNA and Restriction Enzyme Digests
A. DNA fragments (e.g., due to restriction enzyme digestion) can be analyzed by gel
B. Movement of the DNA in the gels (Rf) is inversely proportional to the log(# of base pairs).
C. Polyarcylamide gels are utilized to separate DNA fragments up to approximately 1000
D. Agarose gels are utilized to separate DNA fragments up to about 20,0000 base pairs.
E. DNA bands in gels can be recognized and visualized by...
1. Autoradiography (with32P-labeled DNA fragments).
2. Staining with chemical stains (e.g., methylene blue).
3. Staining with fluorescent probes (e.g., ethidium bromide).
4. Southern blotting followed by autoradiography. (RNA is detected by a similar technique
called Northern blotting.)
IV. DNA Sequencing Via the Maxam-Gilbert Method
A. In the Maxam-Gilbert Method, a single-stranded DNA is labeled at the 5' end with32P (via
B. The DNA strands are broken by specific chemical "cuts" at the 5'-side of each base; one
cut per strand.
C. The resulting DNA fragments are analyzed by polyacrylamide gel electrophoresis and the
DNA sequence is "read" from the gel.
D. Specific DNA cleavage reagents are selected based upon their ability to "cut" in a site
1. DNA (end labeled with32P) is treated with dimethylsulfate, methylating guanine at N-7
and adenine at N-3.
a. Heating the methylated DNA at near neutral pH removes methylated purine (A and G).
b. Subsequent heating in alkali "cuts" at G locations.
c. Subsequent heating in dilute acid "cuts" at both A and G locations.
2. DNA is treated with hydrazine to remove pyrimidines (C and T). Treatment with
hydrazine in the presence of 2 M NaCl removes only C.
a. Subsequent treatment with piperidine "cuts" at both C and T.
b. If 2 M NaCl was present, piperidine "cuts" only at C.
3. Analysis of these four chemical treatments on a polyarylamide gel permits "reading" the
DNA sequence from the gel.
V. DNA Sequencing Via the Sanger Dideoxy Method
A. The Sanger Dideoxy Method utilizes controlled interruption of enzymatic replication.
B. Interruption is accomplished by copying DNA with DNA polymerase I (and appropriate
radioactively labeled primer, dATP, dTTP, dCTP, cGTP, Mg2+) in the presence of selected
1. 2',3'-didexoy analogs have no 3'-OH and, once incorporated in DNA, stop chain
2. A "library" of DNA fragments are produced, all ending at the specific 2',3'-didexoy analog.
3. The four samples - one for each 2',3'-didexoy analog - are run on a polyarcylamide gel and
detected by autoradiography.
4. The DNA sequence is "read" from the autoradiogram.
5. Alternatively, different fluorescent labels may be attached to the primers, and the resulting
pooled DNA fragments detected in the electrophoretic gel by their fluorescence.
VI. Solid-Phase Synthesis of DNA Via the Phosphite Triester Method
A. Solid-phase synthesis of DNA is similar to protein synthesis: activated monomers are are
linked one-at-a-time to a growing chain attached to a solid support.
B. DNA chains of up to 100 nucleotides in length can by synthesized.
C. Protonated dexoyribonucleoside-3'-phosphoramidites (with 5'-OH groups blocked by
DMT [dimethoxytrityl] protecting groups and base amino groups blocked) are joined to the
5'-OH of a growing chain attached to a resin.
D. A coupling cycle consists of...
1. Coupling of the protonated dexoyribonucleoside-3'-phosphoramidite.
2. Oxidation with iodine to form the phosphotriester.
3. Deprotection (removal) of the DMT group by treatment with dichloroacetic acid.
4. Repeating the cycle until last base is added.
5. Treatment with ammonia to remove all protecting groups and cut the oligonucleotide from
6. Hydrolysis of the phosphotriester to form the final oligonucleotide.
E. The solid-phase synthesis allows...
1. Preparation of32P- or fluorescent tag-containing oligonucleotides for hybridization probes.
2. Preparation of specific primers.
3. Synthesis of "tailor-made" genes for an engineered protein.
4. Synthesis of arrays of oligonucleotides for sequencing by hybridization via DNA chips.
VII. DNA Sequencing By Hybridization to Oligonucleotide Arrays (DNA Chips)
A. A high-density array of octamer oligonucleotide sequences is synthesized on a solid
support (a chip) via light-directed synthesis.
B. Fluorescent-labeled oligonucleotide binds to complementary sequences on the chip and is
detected by fluorescence.
C. Sequence homology is determined by location on the chip.
VIII. Utilization of Restriction Enzymes to Form Recombinant DNA Molecules
A. A vector (plasmid or phage) is cut with a restriction enzyme, producing cohesive or
B. The DNA fragment with the desired sequence is treated with the same restriction enzyme.
C. The cut DNA fragment and vector DNA are mixed, annealing at the cohesive ends.
D. The phosphodiester backbone is sealed by DNA ligase.
E. Alternatively, DNA or vector fragments without sites for cleavage by restriction enzymes
can be coupled to chemically synthesized DNA linker (with cut sites) using T4 ligase (a blunt-
IX. Utilization of Plasmids and Lambda Phage as Vectors for DNA Cloning in Bacteria
A. Plasmids are accessory chromosomes that occur naturally in some bacteria.
1. Plasmids are circular, double-stranded DNA.
2. Plasmids vary in size (two thousand to several hundred thousand base pairs).
3. Plasmids carry the genes for...
a. Inactivation of antibiotics.
b. Production of toxins.
c. Breakdown of natural products.
4. A bacteria may have 0-20 copies of a plasmid.
B. pBR322 is one of the more useful plasmids.
1. It contains the genes for tetracycline and ampicillin resistance.
2. It can be cleaved by a variety of restriction endonucleases.
3. EcoR1 cleavage does not affect antibiotic resistance.
4. HindIII, SaII and BamHI cleavage inactivates tetracycline resistance (insertional
C. Lambda phage is probably the most widely used bacteriophage vector.
1. Lambda phage infects bacterial cells and lives in one of two "styles" or "pathways:"
a. Lytic pathway - fully expressed viral activity
b. Lysogenic pathway - phage DNA is inserted into the host genome, but remains inactive.
2. Environmental changes trigger the transition from lysogenic to lytic pathway.
3. Mutant lambda phage have two EcoR1 cleavage sites, used to splice in DNA fragments (up
to 10 kb).
4. Other lambda mutants (e.g., M13) are very useful in DNA sequencing strategies.
X. Cloning Specific Genes From A Genomic Digest
A. Specific DNA sequences can be isolated from the genome using lambda phage.
B. Genomic DNA is fragments (by shearing or by enzymatic digest to 15-20 kb size).
C. Resulting DNA fragments are joined to lambda DNA and packaged in vitro.
D. Recombinant phage are plated on a lawn of E. coli and grown.
E. Infected bacteria are screened by nitrocellulose blot, and probing the nitrocellulose blot
with radioactively labeled probe.
F. Colonies expressing the recombinant lambda phage bind the probe, and may be isolated
and grown in large quantities.
1. The DNA probe may be obtained from mRNA utilizing reverse transcriptase.
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