Molecular Diagnostic Methods Recombinant DNA Technology Recombinant DNA Technology.
Recombinant DNA I
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Transcript of Recombinant DNA I
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Recombinant DNA I
Basics of molecular cloning
Polymerase chain reaction
cDNA clones and screening
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Recombinant DNA Technology
• Utilizes microbiological selection and screening procedures to isolate a gene that represents as little as 1 part in a million of the genetic material in an organism.
• DNA from the organism of interest is divided into small pieces that are then placed into individual cells (usually bacterial).
• These can then be separated as individual colonies on plates, and they can be screened to find the gene of interest.
• This process is also called molecular cloning.
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DNA pieces are joined in vitro to form recombinant molecules
• Generate sticky ends on the DNA, e.g. with restriction endonucleases
• Tie DNA molecules from different sources together with DNA ligase
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Restriction endonucleases generate ends that facilitate mixing and matching
GAATTCCTTAAG
GAATTCCTTAAG
GCTTAA
AATTC G
GCTTAA
AATTC G
EcoRI cut
Mix and ligate
GCTTAAAATTC G
GCTTAAAATTC G
Recombinant molecules
GAATTCCTTAAG
GAATTCCTTAAG
Parental molecules
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DNA ligase covalently joins two DNA molecules
• Uses ATP or NADH to provide energy to seal nicks
P P P
P
P P P P P P P
P P P P P P
P
PP P P
A G G A A T T C G T A
T C C T T A A G C A T
OH
OH
nick
nick
P P PP
P P P P P P P
P P P P P P P
P
P P P
A G G A A T T C G T A
T C C T T A A G C A T
T4 DNA ligase + ATP
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Alternate method to join DNA: homopolymer tails
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Alternate method to join DNA:
linkers
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Introduction of recombinant DNA into living cells via vectors
• Autonomously replicating DNA molecules– (have an origin of replication)
• Selectable marker, such as drug resistance
• Insertion site for foreign DNA– (often a genetically engineered multiple cloning
region with sites for several restriction enzymes)
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Plasmid vectors
• Circular, extrachromosomal, autonomously replicating DNA molecules
• Frequently carry drug resistance genes
• Can be present in MANY copies in the cell
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A common plasmid cloning vector: pUC
ColE1 originof replication
lacZ
mulitplecloning sites
ApR
pUC
ColE1 ori
lacZ
ApR
pUC recombinant
Lac+, or blue colonieson X-gal in appropriatestrains of E. coli
Lac-, or white colonieson X-gal in appropriatestrains of E. coli
foreign DNA
High copy number
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Transformation of E. coli
• E. coli does NOT have a natural system to take up DNA
• Treat with inorganic salts to destabilize cell wall and cell membrane
• During a brief heat shock, some of the bacteria takes up a plasmid molecule
• Can also use electroporation
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Phage vectors
• More efficient introduction of DNA into bacteria
• Lambda phage and P1 phage can carry large fragments of DNA– 20 kb for lambda– 70 to 300 kb for P1
• M13 phage vectors can be used to generate single-stranded DNA
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YAC vectors for cloning large DNA inserts
CEN4ori
URA3
TEL TEL
TRP1
CEN4ori
SUP4
TRP1
URA3TEL TEL
pYAC3
BB
S
Cut with restrictionEnzymes S + B
Ligate to very largeFragments of genomicDNA
Large insert, 400 to as much as 1400 kb
11.4 kb
Yeast artificial chromosome = YAC
Not to scale.
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Bacterial artificial chromosomes
• Are derived from the fertility factor, or F-factor, of E. coli
• Can carry large inserts of foreign DNA, up to 300 kb
• Are low-copy number plasmids
• Are less prone to insert instability than YACs
• Have fewer chimeric inserts (more than one DNA fragment) than YACs
• Extensively used in genome projects
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BAC vectors for large DNA inserts
Cut with restriction enzyme E, remove “stuffer”
Ligate to very large fragments of genomic DNA
SacBII
promoter
oriF
Cm(R)
pBACe3.6
EES
11.5 kb
SacB+: SacBII encodes levansucrase, which converts sucrose to levan, a compound toxic to the bacteria.
Large insert, 300kb
oriFCm(R)
promoter
SSacBII
SacB-: No toxic levan produced on sucrosemedia: positive selection for recombinants.
Not to scale.
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PCR provides access to specific DNA segments
• Polymerase Chain Reaction
• Requires knowledge of the DNA sequence in the region of interest.
• As more sequence information becomes available, the uses of PCR expand.
• With appropriate primers, one can amplify the desired region from even miniscule amounts of DNA.
• Not limited by the distribution of restriction endonuclease cleavage sites.
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Polymerase chain reaction, cycle 1Primer 1 Primer 2
Template
1. Denature
2. Anneal primers
3. Synthesize new DNA with polymerase
Cycle 1
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Polymerase chain reaction, cycle 21. Denature
2. Anneal primers
3. Synthesize new DNA with polymerase
Cycle 2
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PCR, cycle 3
1. Denature
2. Anneal primers
3. Synthesize new DNA with polymerase
Cycle 3 (focus on DNA segments bounded by primers)
2 duplex molecules of desired product
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PCR, cycle 4: exponential increase in product
Cycle 4: Denature, anneal primers, and synthesize new DNA:
6 duplex molecules of desired product
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PCR, cycle 5: exponential increase in product
Cycle 5: Denature, anneal primers, and synthesize new DNA:
14 duplex molecules of desired product
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PCR: make large amounts of a particular sequence
• The number of molecules of the DNA fragment between the primers increases about 2-fold with each cycle.
• For n = number of cycles, the amplification is approximately [2exp(n-1)]-2.
• After 21 cycles, the fragment has been amplified about a million-fold.
• E.g. a sample with 0.1 pg of the target fragment can be amplified to 0.1 microgram
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PCR is one of the most widely used molecular tools in biology
• Molecular genetics - obtain a specific DNA fragment– Test for function, expression, structure, etc.
• Enzymology - place fragment encoding a particular region of a protein in an expression vector
• Population genetics - examine polymorphisms in a population
• Forensics - test whether suspect’s DNA matches DNA extracted from evidence at crime scene
• Etc, etc