Download Recombinant DNA Technology for the non

Recombinant DNA
Technology for the nonscience major.
Historical Perspective
• Pre-history
before 1928
• Ancient
before 1944
• Medieval
1944-1952
• Renaissance
1953-1971
• Modern Era
1971 to present
Further Historical Perspective
Geneticists have known for a long time how to
isolate DNA from cells.
Geneticists have known for a long time how to
chop DNA into small pieces.
What geneticists did not know how to do until the
early 1970s was to replicate small fragments of
DNA.
1970s Breakthrough:
• The discovery of the
restriction enzyme (or
restriction endonuclease).
Properties of RE:
• Cut double-stranded DNA at
specific target sites.
• Allow fragments of DNA that
have been cut with the same RE
to be rejoined.
Properties of RE con’t:
• There are hundreds of
popular RE. They all
recognize a small target
sequence (4-8 b.p.).
The joining of two DNA fragments by DNA
ligase produces a recombinant DNA molecule
Therefore, eukaryotic DNA could be
propagated in prokaryotic cells.
A great breakthrough!!!!!
•
Carriers of foreign DNA are
Vectors:
• Most vectors are derived
from:
• 1. Plasmids
• 2. Bacteriophages
• 3. Cosmids (artificial
constructions)
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• A prokaryotic vector should:
1. Be capable of autonomous replication
independent of the main bacterial chromosome
2. Be easy to isolate, i.e. small.
3. Be non-toxic to host cells.
4. Have space for foreign inserts.
5. Have unique restriction sites for common
restriction enzymes.
6. Have convenient markers for selection of
transformants, e.g. antibiotic resistance genes.
7. Be relaxed, i.e. multiple copies in a host cell.
•A
bacteriophage
is a virus that
infects a
bacteria.
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Introduction to PCR
• PCR (polymerase chain
reaction).
* prc
What is PCR?
• PCR is site-specific in vitro
DNA replication.
DNA Replication Review:
• Add DNA polymerase, all 4 DNA building
blocks  ???
5’ CTGACGCTGCTGCATGCTAGCT 3’
3’ GACTACGACGACGTACGATCGA 5’
DNA Replication Review:
• Primers are required:
5’ CTGACGCTGCTGCATGCTAGCT 3’
CGA 5’
5’ CTG
3’ GACTACGACGACGTACGATCGA 5’
DNA Replication Review:
• Primers are required:
5’ CTGACGCTGCTGCATGCTAGCT 3’
. . . t a c g a t CGA 5’
5’ CTG a t g c t g . . . .
3’ GACTACGACGACGTACGATCGA 5’
Action of DNA
Polymerases:
• DNA polymerases can
add new nucleotides to an
exposed 3’ end!
PCR reaction mix:
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All 4 DNA building blocks (A,C, G, & T)
Taq DNA polymerase (heat resistant)
DNA to be replicated
A pair of primers
Human Chromosome
# 8:
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Human Chromosome
# 8:
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PCR product?
• The binding of the primers determines
where the DNA is replicated.
• The PCR product is a double-stranded
DNA molecule with its ends defined by
the location of primer binding sites.
PCR
• PCR reaction mix incubates for
about 2 hours in a thermocycler
(fancy incubator).
• The thermocycler heats and cools
through 30-40 temperatures cycles.
• DNALC: PCR Animation
Two impt. PCR questions:
• What is the source of a pair of
primers?
What information is a prerequisite
for PCR?
PCR Applications:
#1- Cell-free rapid gene cloning!!
#2- Gene cloning/amplification
from a miniscule sample size.
Introduction to Agarose
Gel Electrophoresis
Weigh out ~ a gram of agarose.
• Mix the agarose
with 50- 100 ml
of buffer.
• Heat to
dissolve
the
agarose.
• Assemble
the gel
tray and
comb.
Pour the gel.
• Pick up the
DNA sample
with a
microliter
pipettor.
• Load one
DNA
sample
into each
well on
the gel.
Connect the gel to a low voltage
power supply.
After completion of the run, add a DNA
staining material and visualize the DNA
under UV light.
• Analyze the
results.
• Gel Electrophoresis
• Electrophoresis
Introduction to
DNA Sequencing
Technology
Dideoxy Sequencing (Sanger
Sequencing, Chain Terminator
method).
• Clone the fragments to be sequenced
into the virus M13.
• Why M13?
• The clones that are isolated are singlestranded DNA.
Primer
^^^
AGCATGATGTCGAGCGAGTCGTACGGT-----
M13
DNA sequencing reaction:
1) DNA fragment to be sequenced cloned into the
vector M13
2) DNA polymerase
3) “Universal” primer
4) All 4 DNA building blocks
5) One ddNTP
The most popular technique is
based on the dideoxynucleotide.
Set up 4 separate reactions. Each
reaction contians one of the 4
ddNTPs. Each ddNTP is tagged
with a radioactive tracer.
A reaction (with ddA)  21, 26, 29, . . . .
T reaction (with ddT)  25, 31, 35, . . . . .
C reaction (with ddC)  22, 23, 27, . . . .
G reaction (with ddG)  ??
Primer (20 nt.)
^^^
AGCATGATGTCGAGCGAGTCGTACGGT-----
M13
• Each reaction generates a set of unique
fragment lengths.
• All fragment lengths are represented.
• None of the fragments are present in
more than one reaction.
• DNA sequencing technology
requires gel electrophoresis
system with the ability to
separate DNA fragments that
separate by one b.p.
DNA sequencing, as performed
in the 1980s (manually) is slow
and labor intensive.
• NCBI HomePage
• DNALC: Cycle Sequencing
• CE Theory
• A Timeline of The Human Genome
• YEAR# human genes mapped to a definite
chromosome location# years it would take to
sequence the human genome
• 1967
none
sequencing not possible yet
• 1977
3 genes mapped
• 4,000,000 years to finish at 1977 rate
• 1987
12 genes mapped
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Organisms sequenced
Year # genomes sequenced
1994 0
1995 2
1996 4
1997 8 (est.)
199830 (est.)
2001 ~75
• DOE Joint Genome Institute