Biology 340 Molecular Biology
Lectures 9, 10 and 11
Recombinant DNA
Feb. 5, 7, and 12, 2001
Reading: Chap. 7 Lodish et al.
Other Assignments: Discussion questions
Outline:
1. Cloning into plasmid vectors
2. Restriction enzymes
3. Lambda libraries
4. Cosmids, BACs, YACs
5. Identifying a gene of interest
6. Characterizing genes
7. Bioinformatics
8. Hybridization methods
9. Methods for quantitating RNA
10. Mapping transcription start sites
11. Protein expression systems
12. In vitro systems
13. Polymerase Chain Reaction
14. DNA Microarrays
Emphasis: Be able to use a correct sequence of methods to solve an
experimental problem.
Lecture: What is recombinant DNA?
Recombinant DNA: DNA chimera from 2 different species, usually a bacterial
plasmid or viral DNA containing a gene from some other species.
Recombinant DNA technology: Variety of methods used for constructing and
characterizing recombinant DNA molecules.
Genetic engineering: Generally, the application of recombinant DNA
techniques.
1. Cloning into plasmid vectors
(Review) Be able to describe the sequence of steps to create a
recombinant DNA in an E. coli plasmid vector and to reintroduce the plasmid
back into bacteria (Fig. 7-3).
A. Isolate DNA
B. Digest DNA with restriction enzymes
C. Link the DNAs with ligase
D. Transform competent E. coli with rDNA
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E. Use selective plating to identify cells containing the rDNA or the
plasmid vector alone.
2. Restriction enzymes
(Review) What are restriction enzymes? What is their role in bacteria
and blue green algae, the organisms they are usually isolated from?
Be able to define the following terms related to the properties of restriction
enzymes: endonuclease, palindromes, staggered ends, blunt ends,
isoschizomers, recognition sequence, compatible cohesive ends, methylases.
3. Lambda libraries
(Review) Lambda is a lysogenic virus that infects E. coli. It can undergo a
lytic life cycle (i. e. infect and lyse its host) or a lysogenic life cycle (i. e. sit
quietly in the bacterial chromosome).
In lambda cloning vectors, the lysogenic genes are replaced by foreign
DNA. Different lambda vectors can be used for constructing either genomic or
cDNA libraries. Recombinant DNAs can be introduced back into E. coli via a
phage (viral) infection.
Be able to distinguish between a genomic and a cDNA library. Be
able to outline the steps involved in constructing a genomic library (Fig. 712) or a cDNA library (Fig. 7-15).
4. Cosmids, BACs, YACs
Cosmids are cloning vectors (~45 kb capacity) that consist of a plasmid
vector containing the cos sites of the lambda virus.
BACs are bacterial artificial chromosomes, high capacity (up to 300 kb)
cloning vectors containing the origin of replication from bacterial F (fertility)
plasmids.
YACs are yeast artificial chromosomes, with capacities of up to 1000 kb.
5. Identifying a gene of interest
A. hybridization
Methods typically involve transferring colonies or plaques to a membrane.
The membrane is incubated with a tagged "probe" (DNA or RNA sequence)
directed toward the gene of interest. The probe forms a hybrid by base pairing to
the DNA sequence attached to the membrane (Fig. 7-18).
B. oligonucleotide probes
If the gene encoding a protein of interest has not been isolated, the amino
acid sequence of the protein can be used to design short (17-30 nt) primers that
can be used to identify the gene by hybridization. "Reverse genetics" is used to
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take the amino acid sequence and predict the nucleotide sequence of a region of
the gene (Fig. 7-19); the short DNA sequence can then be synthesized
chemically.
C. expression of fusion proteins
Antibodies or specific chemical assays can be used to detect the product
of a gene expressed in bacteria, to identify a clone containing the gene of interest
(Fig. 7-21).
6. Characterizing genes
A. gel electrophoresis: Resolves DNA or RNA molecules or fragments
of different sizes. Use agarose gels to separate large fragments, polyacrylamide
gels to separate small fragments.
B. restriction mapping: Restriction sites serve as the "landmarks" along
a stretch of DNA. Sites can be mapped using techniques that end-label the DNA
(Fig. 7-25) or detect unlabeled DNA fragments.
C. pulsed field electrophoresis: Electrophoresis in a field that is turned
on and off at short intervals. Used to separate large DNA fragments and small
(yeast) chromosomes.
D. DNA sequencing
--Maxam and Gilbert method: Early method which relies on different
susceptibilities of the bases on DNA to chemical cleavage to generate fragments
of different lengths. Fragments are separated on a denaturing polyacrylamide
gel (Fig. 7-27).
--dideoxy (Sanger): Most widely used method that has been modified for
high capacity sequencing. Involves enzymatic synthesis of DNA fragments of
different lengths that are separated on a denaturing gel (Fig. 7-29 and CD-rom).
7. Bioinformatics
Use of computers to collect, organize and analyze DNA and protein
sequence data.
8. Hybridization methods
A. Southern Blot--Detection of a particular DNA sequence on a
membrane filter using a tagged DNA or RNA probe (Fig. 7-32). Used to map a
gene on a chromosome, to locate the site in the genome of a particular gene, or
to compare the relatedness of genes from different species.
B. Northern Blot--Detection of a particular RNA sequence on a
membrane filter using a tagged DNA or RNA probe. Used to identify the tissues
or cells that express a particular RNA.
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9. Methods for quantitating RNA
A. RNase protection: Can be used to determine the amount of a
specific RNA in a mixture. Hybridization of an RNA to a labeled, single-stranded
probe protects it from digestion by a single-strand specific nuclease. The size of
the protected region can be determined by gel electrophoresis. See Fig. 7-34.
B. RT-PCR: Reverse transcriptase PCR is a modification of the
polymerase chain reaction in which a DNA copy is first made from RNA by
reverse transcriptase, then the reaction is subjected to PCR. To use for
quantitating RNA, must have an internal control.
10. Mapping transcription start sites (see Fig. 7-35)
A. Primer extension: Use of a primer, the mRNA for a gene and reverse
transcriptase to synthesize a product. The primer extension product is used to
map the start site.
B. S1 protection: A probe which extends beyond the start site is
annealed to the mRNA and digested with S1 nuclease (a single-strand specific
nuclease). The size of the product is used to map the start site.
11. Protein expression systems
The following vectors are used in expressing large amounts of
recombinant proteins in E. coli.
A. LacZ vectors: Contain the gene for the enzyme -galactosidase.
There are many variations of vectors that contain this gene and the regulatory
elements (from the lactose operon) that control it. The text describes a vector in
which the lacZ gene can be replaced by another gene, which would be under
inducible control. In other vectors, the lacZ gene can be disrupted for easy
selection of recombinants.
B. T7 vectors (pET vectors): Use viral T7 RNA polymerase promoter
under inducible control to express high levels of foreign proteins in E. coli (Fig. 737).
C. GST vectors: Allow cloning downstream of the glutathione-Stransferase gene to produce recombinant fusion proteins in E. coli.
12. In vitro systems
A. transcription: Vectors containing genetically engineered promoters
recognized by viral RNA polymerases (T7, T3 or Sp6) enable synthesis of
transcripts or labeled RNA probes in a test tube.
B. translation: Cell extracts from rabbit reticulocytes or wheat germ can
be used to synthesize proteins from mRNA added to a test tube.
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13. Polymerase Chain Reaction
Method of amplifying DNA using a thermostable DNA polymerase, primers
and a small amount of template DNA. See Fig. 7-38 and CD-rom. Many
applications, including cloning, forensics, DNA sequencing and quantitating
mRNAs.
14. DNA Microarrays
Essentially cDNA or genomic libraries or individual oligonucleotides
spotted by the hundreds or thousands on membranes or silica slides. Can be
hybridized to different populations of fluorescently tagged cDNAs to quantitate
differences in gene expression (Fig. 7-39). May be used in the future for tailoring
drug design to individual patients.
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