recumbinant DNA.pdf

Recombinant DNA Technology and Other Molecular Methods
Cloning technology = generation of many copies of DNA (e.g.,
recombinant DNA) by replication in a host.
✓ Developed in the 1970s.
✓ Can generate un unlimited supply of gene copies.
✓ Many applications:
• Genetic mapping
• DNA sequencing
• Mutation studies
• Transformation
• Genetic engineering
Recombinant DNA Libraries
Restriction Enzyme Site Analysis
Analysis of Gene Transcripts (What is being expressed?)*
Polymerase Chain Reaction (PCR) & DNA sequencing
(to be included with chapter 8 lecture topics)
Genetics from mendale to microchip
array Molecular Genetics

DNA Cloning (Overview)
Goal is to generate large amounts of pure DNA that can be
manipulated and studied.
DNA is cloned by the following steps:
1. Isolate DNA from organism (e.g., extract DNA)
2. Cut DNA with restriction enzymes to a desired size.
3. Splice (or ligate) each piece of DNA into a cloning vector to create
a recombinant DNA molecule.
Cloning vector = artificial DNA molecule capable of replicating
in a host organism (e.g., bacteria).
4. Transform recombinant DNA (cloning vector + DNA fragment)
into a host that will replicate and transfer copies to progeny.

Step 1-Isolate whole genomic DNA from organism
DNA extraction easily performed using:
• SDS (detergent) to break up cell membrane and organelles.
• Salt (NaCl) lyses cells and binds the DNA strands together.
• Proteinase K to digest proteins bound to DNA.
• Ethanol (EtOH) to precipitate and wash DNA.
• Water to resuspend and store DNA.
Storage - DNA can be stored short-term at room temperature,
but is best stored long-term at -80C or in liquid nitrogen.
Ideal storage conditions - large uncut DNA fragments.
Suboptimal storage conditions - many small DNA fragments.
*Average size of DNA fragments is important for applications
involving large regions of DNA sequence/less important for
applications involving short regions of DNA sequence.

Step 2-Cut DNA with restriction enzymes
Restriction enzymes recognize specific bases pair sequences in DNA
called restriction sites and cleave the DNA by hydrolyzing the
phosphodiester bond.
✓ Cut occurs between the 3’ carbon of the first nucleotide and the
phosphate of the next nucleotide.
✓ Restriction fragment ends have 5’ phosphates & 3’ hydroxyls.
Fig. 3.4
restriction
enzyme

Step 2-Cut DNA with restriction enzymes (cont.)
✓ Most restriction enzymes occur naturally in bacteria.
✓ Protect bacteria against viruses by cutting up viral DNA.
✓ Bacteria protects their DNA by modifying possible restriction sites
(methylation).
✓ More than 400 restriction enzymes have been isolated.
✓ Names typically begin with 3 italicized letters.
Enzyme Source
EcoRI E. coli RY13
HindIII Haemophilus influenzae Rd
BamHI Bacillus amyloliquefaciens H
✓ Many restriction sites are palindromes of 4-, 6-, or 8-base pairs.
✓ Short restriction site sequences occur more frequently in the
genome than longer restriction site sequences, e.g., (1/4)n.

, EcoRI
“6-base cutter”

Step 2-Cut DNA with
restriction enzymes
(cont.)
✓ Some restriction
enzymes produce blunt
ends, whereas others
produce sticky
(overhanging
staggered) ends.
✓ Sticky ends are useful
for DNA cloning
because complementary
sequences anneal and
can be joined directly
by DNA ligase.
Fig. 7.2

Cut and ligate 2 DNAs with EcoRI ---> recombinant DNA

Step 3-Splice (or ligate) DNA into a cloning vector to create a
recombinant DNA molecule
Six different types of cloning vectors:
1. Plasmid cloning vectors
2. Phage  cloning vectors
3. Cosmid cloning vectors
4. Shuttle vectors
5. Yeast artificial chromosomes (YACs)
6. Bacterial artificial chromosomes (BACs)

Plasmid Cloning Vectors:
✓ Bacterial plasmids, naturally occurring, circular double-stranded
extrachromosomal DNA elements capable of replicating
autonomously within a cell.
✓ Plasmid vectors engineered from bacterial plasmids for use in
cloning.
✓ Required features (e.g., E. coli plasmid vectors):
1. Origin sequence (ori) required for replication.
2. Selectable trait that enables E. coli that carry the plasmid to
be separated from E. coli that do not (e.g., antibiotic
resistance, grow cells on antibiotic; only those cells with the
anti-biotic resistance grow in colony).
3. Unique restriction site such that an enzyme cuts the plasmid
DNA only once. A fragment of DNA cut with the same
enzyme can then be inserted into the plasmid restriction site.
4. Simple marker that allows you to distinguish plasmids that
contain inserts from those that do not (e.g., lacZ+ gene)

pUC19
Polylinker:
restriction
sites
Origin
sequence
Ampicillin
resistance
gene
lacZ+
gene

Some features of pUC19:
1. High copy number in E. coli, ~100 copies/cell, provides high yield.
2. Selectable marker is ampR. Ampicillin in growth medium prevents
growth of all other E. coli.
3. Cluster of restriction sites called a polylinker occurs in the lacZ (-
galactosidase) gene.
4. Cloned DNA disrupts reading frame and -galactosidase production.
5. Add X-gal to medium; turns blue in presence of -galactosidase.
6. Plaque growth: blue = no inserted DNA and white = inserted DNA.
7. Some % of digested vectors will reanneal with no insert. Remove
5’ phosphates with alkaline phosphatase to prevent
recircularization (this also eliminates some blue plaques).
8. Plasmids are transformed into E. coli by chemical incubation or
electroporation (electrical shock disrupts the cell membrane).
9. Cloned inserts >5-10 kb typically are unstable; good for <10kb.

Fig. 7.5
*Cut with same
restriction enzyme
*DNA ligase

Phage  cloning vectors:
1. Engineered version of bacteriophage  (infects E. coli).
2. Central region of the  chromosome (linear) is cut with a
restriction enzyme and digested DNA is inserted.
3. DNA is packaged in phage heads to form virus particles.
4. Phages with both ends of the  chormosome and a 37-52 kb
insert replicate by infecting E. coli.
5. Phages replicate using E. coli and the lytic cycle (see Fig. 3.13).
6. Produces large quantities of 37-52 kb cloned DNA.
7. Like plasmid vectors, large number of restriction sites available;
useful for larger DNA fragments than pUC19.

Cosmid cloning vectors:
1. Features of both plasmid and phage cloning vectors.
2. Do not occur naturally; circular.
3. Origin (ori) sequence for E. coli.
4. Selectable marker, e.g. ampR.
5. Restriction sites.
6. Phage  cos site permits packaging into  phages and
introduction to E. coli cells.
7. Useful for 37-52 kb.

Shuttle vectors:
1. Capable of replicating in two or more types of hosts..
2. Replicate autonomously, or integrate into the host genome and
replicate when the host replicates.
3. Commonly used for transporting genes from one organism to
another (i.e., transforming animal and plant cells).
Example:
*Insert firefly luciferase gene
into plasmid and transform
Agrobacterium.
*Grow Agrobacterium in large
quantities and infect tobacco plant.

Yeast Artificial Chromosomes (YACs):
Vectors that enable artificial chromosomes to be created and cloned
into yeast.
Features:
1. Yeast telomere at each end.
2. Yeast centromere sequence.
3. Selectable marker (amino acid dependence, etc.) on each arm.
4. Autonomously replicating sequence (ARS) for replication.
5. Restriction sites (for DNA ligation).
6. Useful for cloning very large DNA fragments up to 500 kb; useful
for very large DNA fragments.

Bacterial Artificial Chromosomes (BACs):
Vectors that enable artificial chromosomes to be created and cloned
into E. coli.
Features:
1. Useful for cloning up to 200 kb, but can be handled like regular
bacterial plasmid vectors.
2. Useful for sequencing large stretches of chromosomal DNA;
frequently used in genome sequencing projects.
3. Like other vectors, BACs contain:
1. Origin (ori) sequence derived from an E. coli plasmid called
the F factor.
2. Multiple cloning sites (restriction sites).
3. Selectable markers (antibiotic resistance).

Recombinant DNA Libraries (3 types):
1. Genomic library, Collection of cloned restriction enzyme digested
DNAs containing at least one copy of every DNA sequence in a
genome.
2. Chromosome library, Collection of cloned restriction enzyme
digested fragments from individual chromosomes.
3. Complementary DNA (cDNA) library, Collection of clones of DNA
copies made from mRNA isolated from cells.
✓ reverse transcriptase (RNA dependent DNA polymerase)
✓ Synthesizes DNA from an RNA template
✓ cDNA libraries reflect what is being expressed in cells.
✓ # of clones required for a complete library can be calculated
from the size of the genome and average size of overlapping
fragments cut by restriction enzymes.
✓ Library should contain many times more clones than the
calculated minimum number of clones.

Genomic Library:
3 ways to make a genomic library:
1. Complete digestion (at all relevant restriction sites)
1. Produces a large number of short DNA clones.
2. Genes containing two or more restriction sites may be cloned
in two or more pieces.
2. Mechanical shearing
1. Produces longer DNA fragments.
2. Ends are not uniform, requires enzymatic modification before
fragments can be inserted into a cloning vector.
3. Partial digestion
1. Cut at a less frequent restriction site and limit the amount and
time the enzyme is active.
2. Results in population of large overlapping fragments.
3. Fragments can be size selected by agarose electrophoresis.
4. Fragments have sticky ends and can be cloned directly.

Partial digestion with
Sau3A
Results in a library of
overlapping DNA
fragments of various
sizes.

Screening a genomic library (plasmid or cosmid):
1. Plasmid vectors containing digested genomic DNA are transformed
into E. coli and plated on selective medium (e.g., ampicillin).
2. Colonies that grow are then are replicated onto a membrane (E.
coli continues to grow on the membrane).
3. Bacteria are lysed and DNA is denatured.
4. Membrane bound DNA is next probed with complementary DNA
(e.g., 32P radio-labeled DNA).
5. Complementary DNA in the probe is composed of DNA sequence
you are looking for; homologous sequence presumably also found
in library.
6. Unbound probe DNA is washed off the filter.
7. Hybridization of probed DNA is detected by exposure to X-ray film
(or by chemiluminescence).
8. Pattern is noted from exposure pattern of clones on X-ray film.
9. Select clones that test positive and isolate for further analysis.

Screening a genomic
library

Chromosome Library:
1. Screening can be reduced if target genes can be localized to a
particular chromosome.
2. Chromosomes can be separated by flow cytometry.
1. Condensed chromosomes are stained with fluorescent dye.
2. Chromosomes separate based on the level of binding of the
dye and are detected with a laser.

cDNA Library:
1. cDNA is derived from mature mRNA, does not include introns.
2. cDNA may contain less information than the coding region.
3. cDNA library reflects gene activity of a cell at the time mRNAs are
isolated (varies from tissue to tissue and with time).
4. mRNA degrades quickly after cell death, and typically requires
immediate isolation (cryoprotectants can increase yield if
immediate freezing is complicated by field work).
5. Creating a cDNA library:
1. Isolate mRNA
2. Synthesize cDNA
3. Clone cDNA

Creating a cDNA library, Step 1-Isolate mRNA:
• Mature eukaryote mRNA has a poly-A tail at the 3’ end.
• mRNA is isolated by passing cell lysate over a poly-T column
composed of oligo dTs (deoxythymidylic acid) .
• Poly-A tails stick to the oligo dTs and mRNAs are retained, all
other molecules pass through the column.

Creating a cDNA library, Step 2-cDNA synthesis:
3-steps:
• Anneal a short oligo dT (TTTTTT) primer to the poly-A tail.
• Primer is extended by reverse transcriptase 5’ to 3’ creating a
mRNA-DNA hybrid.
• mRNA is next degraded by Rnase H, but leaving small RNA
fragments intact to be used as primers.
• DNA polymerase I synthesizes new DNA 5’ to 3’ and removes
the RNA primers.
• DNA ligase connects the DNA fragments.
• Resultis a double-stranded cDNA copy of the mRNA.

,
Synthesis
of cDNA

Creating a cDNA library, Step 3-How to clone cDNA:
1. cDNA has blunt ends, thus need to add restriction site linkers or
adapters to make them “sticky”.
2. Use T4 DNA ligase and blunt end ligation to add restriction site
linkers (or adapters) to each end of the cDNA.
3. Next, digest the linkers with the same restriction enzyme used
to cleave the vector.
4. Mix cDNA with cut vector DNA in the presence of DNA ligase.
5. Transform into an E. coli host for cloning.
6. If cDNA has the same restriction site as the linkers, cDNA will
be cloned in pieces.
7. Solution, use adapters with single-stranded overhangs that
match the restriction site on the vector.

, Cloning of cDNA using
BamHI linkers
Alternative: use adapter
5’-GATCCAGAC-3’
GTCTG-5’
Genetics from Mendale to microchip

Screening a cDNA library:
1. cDNA libraries are most often used to detect genes for proteins
(cDNAs are generated for genes that are transcribed!).
2. If you know the DNA sequence for the protein coding gene you
want to find, a homologous DNA probe can be used.
3. If no homologous DNA sequence is available, cDNA can be probed
with with an antibody that recognizes the protein.
4. Expression vector: cloned cDNA is inserted between a promoter
and transcription terminator before it is transformed.
5. mRNA is transcribed from the cDNA and translated.
6. Colonies (now expressing proteins) are transferred to membrane.
7. Membrane is incubated with radioactive labeled antibody probe
that recognizes the protein (non-radioactive chemiluminescent
probes also are available).
8. Colonies with bound antibodies leave a dark spot on X-ray film.

Screening a cDNA library
with antibody probe.

Screening a cDNA library (cont.):
1. Identified cDNA can be used to:
1. Isolate and sequence the gene for the protein (either with out
without DNA sequence information).
2. Identify and compare homologous sequences in other types of
organisms.
3. Quantify amount of mRNA synthesized from a gene and
measure the level of gene expression.
2. Synthetic oligonucleotides might be used to probe the library if
DNA sequences are available (degeneracy of the code may require
a mix of different oligonucleotides).
3. Complete cloned cDNA from other organisms showing a high level
of homology also can be used as probes, heterologous probes.

Analysis of Restriction Enzyme Sites:
1. Restriction sites (where restriction enzymes cut DNA sequences)
can be mapped by cutting DNA with selected restriction enzymes,
electrophoresing the DNA on an agarose gel, and visualizing the
DNA banding pattern with ethidium bromide.
2. Mapping the positions of restrictions sites generates a linkage map
that can have many applications: e.g., forensics, systematics,
population genetics.
3. DNA is cut with different enzymes, and each DNA-enzyme mixture
is loaded on a separate lane in the gel.
4. Negatively charged DNAs separate by size in the electric field
(smaller fragments move faster than larger fragments).
5. Fragment pattern produced by gel stained with ethidium bromide
is photographed or imaged and processed by software.
6. Distance each band migrates is calibrated with known size
standards of pre-cut DNA (i.e., ladder).
7. Resulting pattern of different numbers and sizes of fragments cut
by different enzymes is interpreted to make a restriction site map.

Uncut EcoRI BamHI
EcoRI
+
BamHI
5.0 kb 4.5 kb 3.0 kb 2.5 kb
0.5 kb 2.0 kb 2.0 kb
0.5 kb
Small bands - migrate faster
Large bands - migrate slower
0.5 2.5 2.0
BamHI
EcoRI

Applications of Restriction Enzyme Site Analysis:
1. Analyze gene structure with large and fine scale mapping;
limited by number of restriction sites in the target
sequence and the variety of enzymes that are selected for
analysis.
2. Restriction Fragment Length Polymorphism (RFLPs) can be
used for forensic and phylogenetic analyses (e.g., each
individual possess a different pattern due to variation in
number and composition of different restriction sites).
3. Less expensive that DNA sequencing/other genotyping
methods.
4. Cut DNAs can be probed without cloning using the Southern
Blot, which can be used to determine if a gene shares
homology with a probe and how many copies of a gene
might exist.

Southern Blot:
• Invented by Edward Southern.
• After restriction enzyme cutting and electrophoresis, genomic DNA
appears as a continuous smear on agarose gel.
• Denature DNA in the gel with an alkaline solution.
• Neutralize gel and place on blotting paper that spans a glass plate
and reaches into buffer solution.
• Place a Hybridization membrane over the gel, and stack paper
towels and a weight on top of the membrane.
• Buffer solution is wicked up by blotting paper to the paper towels,
passing through the gel and transferring DNA to membrane.
• Fragments on the filter are arranged the same as on the gel.
• Saturate the membrane with a labeled probe (radioactive or
nonradioactive) and expose to x-ray film.

Southern Blot
Components:
(top to bottom)
Weight
Paper towels
Membrane
Gel
Blotting paper
Glass plate
Tray with buffer

Northern Blot:
• Similar to the Southern Blot, but used to probe RNA (not DNA).
• Can be used to determine mRNA size, e.g., detect differences in
the promoter and terminator sites, etc.
• Can be used to determine if a particular gene is expressed, and if
so, how much, what tissue type, and when in the life cycle?
Example:
Expression sybII gene
at different life stages in
the frog Xenopus laevis

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