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
Recombinant DNA I
an organism.
• DNA from the organism of interest is divided into
Basics of molecular cloning small pieces that are then placed into individual
cells (usually bacterial).
Polymerase chain reaction
• These can then be separated as individual
cDNA clones and screening colonies on plates, and they can be screened to
find the gene of interest.
• This process is also called molecular cloning.

Restriction endonucleases generate ends

DNA pieces are joined in vitro to form
that facilitate mixing and matching
recombinant molecules
GAATTC GAATTC

• Generate sticky ends on the DNA, e.g. with CTTAAG

EcoRI cut
CTTAAG

restriction endonucleases G
CTTAA
AATTC
G
G
CTTAA
AATTC
G

• Tie DNA molecules from different sources Mix and ligate

together with DNA ligase

G AATTC
CTTAA G Recombinant
molecules
G AATTC
CTTAA G

GAATTC
CTTAAG Parental
molecules
GAATTC
CTTAAG

Alternate method to join DNA:

DNA ligase covalently joins two DNA molecules
homopolymer tails
• Uses ATP or NADH to provide energy to seal
nicks nick

P P P OH 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
P P P P P P P P P P
OH

P
nick

T4 DNA ligase + ATP

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
P P P P P P P P P P P

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 Introduction of recombinant DNA into
Alternate
living cells via vectors
method to
join DNA: • Autonomously replicating DNA molecules
linkers – (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)

Plasmid vectors A common plasmid cloning vector: pUC

mulitple
cloning
lacZ sites
• Circular, extrachromosomal, autonomously Lac+, or blue colonies
on X-gal in
replicating DNA molecules pUC
appropriate
strains of E. coli
• Frequently carry drug resistance genes
ApR ColE1 origin High copy
• Can be present in MANY copies in the cell of replication number

lacZ foreign DNA

Lac-, or white colonies

pUC recombinant on X-gal in
appropriate
strains of E. coli

ApR ColE1 ori

Transformation of E. coli Phage vectors

• More efficient introduction of DNA into
• E. coli does NOT have a natural system to bacteria
take up DNA • Lambda phage and P1 phage can carry
• Treat with inorganic salts to destabilize cell large fragments of DNA
wall and cell membrane – 20 kb for lambda
• During a brief heat shock, some of the – 70 to 300 kb for P1
bacteria takes up a plasmid molecule • M13 phage vectors can be used to generate
• Can also use electroporation single-stranded DNA

2
 YAC vectors for cloning large DNA inserts Bacterial artificial chromosomes
Yeast artificial chromosome = YAC • Are derived from the fertility factor, or F-
CEN4 SUP4
ori
URA3
factor, of E. coli
TRP1 S
pYAC3 • Can carry large inserts of foreign DNA, up to
Cut with restriction Ligate to very large 300 kb
TEL TEL Enzymes S + B Fragments of genomic
B B
11.4 kb DNA • Are low-copy number plasmids
• Are less prone to insert instability than
TEL TRP1 ori CEN4 URA3 TEL
YACs
• Have fewer chimeric inserts (more than one
Large insert, 400 to
as much as 1400 kb
DNA fragment) than YACs
Not to scale. • Extensively used in genome projects

BAC vectors for large DNA inserts PCR provides access to specific DNA segments
promoter
Cm(R)
S E E SacB+: SacBII encodes levansucrase, • Polymerase Chain Reaction
which converts sucrose to levan,

pBACe3.6
SacBII a compound toxic to the bacteria. • Requires knowledge of the DNA sequence
oriF 11.5 kb in the region of interest.
Cut with restriction enzyme E, remove “stuffer” • As more sequence information becomes
Ligate to very large fragments of genomic DNA
available, the uses of PCR expand.
• With appropriate primers, one can amplify
promoter Large insert, 300kb
SacBII the desired region from even miniscule
S
amounts of DNA.
Cm(R)
oriF SacB-: No toxic levan produced on sucrose • Not limited by the distribution of restriction
media: positive selection for recombinants.
endonuclease cleavage sites.
Not to scale.

Polymerase chain reaction, cycle 1 Polymerase chain reaction, cycle 2

Primer 1 Primer 2
Template Cycle 2 1. Denature

Cycle 1 1. Denature

2. Anneal primers

2. Anneal primers

3. Synthesize new DNA with polymerase

3. Synthesize new DNA with polymerase

3
 PCR, cycle 3 PCR, cycle 4: exponential increase in
Cycle 3 (focus on DNA segments bounded by primers) product
1. Denature
Cycle 4: Denature, anneal primers, and synthesize new DNA:

6 duplex
2. Anneal primers molecules
of desired
product

3. Synthesize new DNA with polymerase

2 duplex
molecules
of desired
product

PCR, cycle 5: exponential increase in PCR: make large amounts of a

product particular sequence
Cycle 5: Denature, anneal primers, and synthesize new DNA:
• The number of molecules of the DNA
fragment between the primers increases
14 duplex
molecules
about 2-fold with each cycle.
of desired • For n = number of cycles, the amplification
product
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

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