I.

Introduction
A. Recombinant DNA technology's success is due to a variety of factors including...
1. Enzymes such as...
a. Restriction enzymes.
b. DNA ligases.
c. DNA and RNA polymerases.
d. Reverse transcriptase.
2. Base-pairing of DNA to its complement DNA (or RNA).
3. The existence of viruses and plasmids and a detailed knowledge of their biochemistry.

B. In this chapter we will examine a variety of recombinant DNA technologies.

II. Restriction Enzymes

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
electrophoresis.
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
base pairs.
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 (with 32P-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 with 32P (via
polynucleotide kinase).
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
specific manner.
1. DNA (end labeled with 32P) 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
2',3'-dideoxy analogs.

1. 2',3'-didexoy analogs have no 3'-OH and, once incorporated in DNA, stop chain
elongation.
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
the resin.
6. Hydrolysis of the phosphotriester to form the final oligonucleotide.

E. The solid-phase synthesis allows...

1. Preparation of 32P- 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.
D. DNA chips have been synthesized containing all 65,536 possible octamers on a single chip
the size of a thumbnail.

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
"sticky" ends.
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-
ended ligase).

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
inactivation).

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.
2. The DNA probes may synthesized from a corresponding protein amino acid sequence.

XI. Cloning of Large Genes Via Chromosome Walking

A. Large DNA sequences (100-1000 kb) can be propagated in yeast artificial chromosomes
(YACs).
B. YACs contain...
1. A centromere,
2. An autonomous replication sequence (ARS), where replication begins,
3. A pair of telomeres, selectable marker genes and a cloning site.
C. Genomic DNA is fragmented and isolated (~450 kb fragments) and cloned into the YAC.
D. Cloned YAC is added to yeast cultures and taken up by the yeast.
E. Recombinant YAC is replicated in the yeast by "normal" mitosis.
F. Base sequences in the YAC can be analyzed by gene walking. In gene walking...
1. A specific DNA fragment from the YAC is detected by hybridization with a probe.
2. Regions on the detected DNA fragment (outside the portion detected by the probe) may be
used as additional probes to detect other DNA fragments.
3. Piecing together of fragments is used to determine overall sequence.

XII. Amplifying (Copying) DNA By the Polymerase Chain Reaction (PCR)

A. PCR was devised in 1984 by Kary Mullis.
B. Technique requires knowledge of flanking sequences on the double stranded DNA at both
ends of the DNA sequence to be copies - one flanking sequence on one strand, one flanking
sequence on the other.

C. A PCR "cycle" consists of...

1. DNA strand separation by heating to 95oC for 15 seconds.
2. Addition of primers (20-30 nucleotides long) for each of the known flanking regions and
abrupt cooling to 54oC to anneal primers.
3. Heating to 72oC for DNA coping by Taq DNA polymerase for approximately 30 seconds.
4. Repeat cycle at step #1.
C. The number of copies (desired DNA sequence plus two flanking sequences) grows
exponentially (million-fold at 20 cycles; billion fold at 30 cycles).
D. Many forensic applications are possible. Potentially, a single DNA molecule can be
amplified and analyzed.

XIII. Preparation of cDNA from mRNA Expressed in Host Cells

A. mRNA is isolated from cells (via poly-T agarose chromatography).
B. cDNA is made from the isolated mRNA using reverse transcriptase and appropriate
dNTPs.
C. The mRNA is digested via alkali treatment.

D. The single-stranded cDNA forms a hairpin loop with itself that primes copying the strand
with DNA polymerase I.
E. The cDNA strand is copied back to the poly-T end, and subsequently treated with S1
nuclease to cut out the hairpin loop.
F. The duplex cDNA can be treated with restriction endonucleases, inserted into an
expression vector (plasmid engineered with a strong promoter and appropriate insertion sites),
and used to infect bacteria.
G. Infected bacterial colonies can be screened on agar plates by a nitrocellulose assay,
looking for the expressed protein (via radiolabeled antibody to the protein).
H. Infected bacterial culture can be used to mass produce the protein (and DNA sequence) of
interest.

XIV. Introduction of New Genes into Eukaryotic Cells

A. Recombinant DNA can be introduced into eukaryotic cells by precipitation with calcium
phosphate.
B. Recombinant DNA can be introduced into eukaryotic cells by microinjection.
C. Retroviruses (RNA tumor viruses) can be used a vectors in eukaryotic cells.

D. Transgenic animals have been developed by injecting microinjecting plasmids into the
pronucleus of fertilized eggs.
1. Injected eggs are placed in the mother's uterus and develop.
2. Offspring that incorporated the DNA into the genome may have multiple copies of the
DNA and express the gene.

XV. Site-Specific Mutagenesis in Protein Engineering

A. New genes to make "designed" proteins can be made by...
1. Deletions,
2. Insertions, or
3. Substitutions.

B. Deletions may be made by...

1. Cleaving plasmids at two sites and reannealing to a smaller plasmid ring size.
2. Cleaving plasmids at one site and using exonucleases to remove a few bases near the cut
prior to reannealing the ring.

C. Substitutions may be made by oligonucleotide-directed mutagenesis.

1. A oligonucleotide (as little as 15 base pairs with one substituted base) with the base
substitution is synthesized and annealed as a primer to the plasmid with the incorporated gene
sequence.
2. Duplex DNA is made, and half of the daughter strands will have the substitution.
3. Expression of the plasmid into protein will produce the mutated protein.

D. Cassette mutagenesis may also be used to engineer proteins.

1. A plasmid with a unique pair of cleavage sites (approximately 40 kb apart) is used as a
vector.
2. A synthetic duplex DNA containing the same cleavage sites is synthesized, cut and inserted
into the plasmid vector.
3. The vector is then put into a bacterium and expressed as a mutated protein.

XVI. General Approaches to Problem Solving Using Recombinant DNA Technology

A. Techniques of protein chemistry and nucleic acid chemistry mutually reinforce each other
in problem solving.
B. Knowledge of a gene or purified protein's amino acid sequence is enough to "problem
solve."