Survey of

molecular techniques

Enzymes for DNA manipulation

• DNA polymerases
• nucleases
• ligases
• end-modification enzymes
 DNA polymerases
(template-dependent)

• synthesize new DNA chains (complementary to template)

• require oligonucleotide primers

• may possess 3’-5’ exonuclease activity (for proofreading)

• some forms are thermostable (e.g., Taq polymerase)

• some forms require RNA as template (RNA polymerases),

synthesizing cDNAs

Nucleases

• hydrolyze phosphodiester bonds in nucleic acids

• some are specific for DNA, others for RNA

• main types:

• exonuclease

• endonuclease
 Exonucleases: examples

• Exonuclease III - removes residues from 3’ ends

of a DNA strand

• Bacteriophage λ exonuclease - removes residues

from 5’ ends of a DNA strand

Endonucleases

• cut at internal phosphodiester bonds

• restriction enzymes - an important class of

DNA endonucleases; play a central role in
recombinant DNA technology
 Restriction enzymes

• Molecular scissors that cut double stranded

DNA at defined sites

• 3 classes
– Type II restriction enzymes are most commonly
used; cut DNA at defined sites

Nomenclature

• Each restriction enzyme is named after the

bacteria it was isolated from

• For example:
EcoRI (pronounced “eeko-are-one”)
E = Genus, Escherichia
co = Species, coli
R = Strain, RY13
I = First endonuclease isolated from
this bacteria
 Restriction Sites
• Specific sequence of nucleotides that a
particular restriction enzyme recognizes
• Most restriction sites are about 4-6 bases
long
• Many are palindromic
- GGATCC -
- CCTAGG -
• Frequency that a restriction site is likely to
occur in a strand of DNA can be predicted
– 1/4n where n = # of bases in the restriction site
– A four base pair restriction site will occur every
256 bases

Types of ends after restriction

 Restriction enzymes: examples

• EcoRI GAATTC sticky (4bp)

• HindIII AAGCTT sticky (4bp)
• Sau3A GATC sticky (4bp)
• PvuII CAGCTG blunt
• HaeIII GGCC blunt
• NotI GCGGCCGC sticky (4bp)
• HinfI GANTC sticky (3bp)

DNA ligases

• catalyze joining of two DNA strands by

synthesizing a phosphodiester bond
 Ligation of DNA fragments
5’ G A A T T C
+
3’ C T T A A G

one DNA fragment another DNA fragment

annealing of sticky ends

5’ G A A T T C 3’

3’ C T T A A G 5’

DNA ligase-catalyzed ligation

G A A T T C

C T T A A G

Ligation of DNA fragments

Ligation of joining of double strands vs.

ligation of nicked fragments
 Recombinant DNA Technology

• Involves the combination of DNA from two different

sources
• Allows researchers to artificially manipulate genetic
material; sequences are cut with restriction enzymes
and joined together with ligases
• Revolutionized the way DNA sequences were studied
– Produce proteins
– Study gene expression
– Gene function

Recombinant Plasmid

+
 End-modification enzymes

• terminal transferase
adds homopolymer tails to the 3’ ends of a linear
duplex
• T4 polynucleotide kinase
adds a phosphate group to the 5’-OH end of a
polynucleotide (to label it or to permit ligation)
• alkaline phosphatase
removes phosphate group from 5’ ends

Polymerase Chain Reaction

(PCR)
 Polymerase Chain Reaction (PCR)

Parameters of a PCR cycle:

an example

denaturation
oC
94 oC extension
1 min 50 oC 72 oC
20 s 1 min
annealing
of primers

DNA
n
 Thermal cycler:
The PCR machine

Agarose gel electrophoresis

Gel: agarose, 0.7 - 2.5 %

+
 Ethidium bromide
as a fluorescent stain

Agarose gel electrophoresis: analysis

size
marker

• Detection of DNA
fragment

• determining sizes of
fragments
 Fidelity of DNA polymerases

Optimization of PCR

• Duration of annealing and elongation steps

• temperature
• Mg++ concentration
• primer concentration
• template concentration
• hot start (wax, Mg++ , antibodies)
• touchdown PCR
• additives (DMSO, betaine)
Variations of PCR

• nested PCR
• inverse PCR
• panhandle PCR
• quantitative PCR
• multiplex PCR
• asymmetric PCR
• in situ PCR
• etc.

cDNA
Isolation of mRNA by affinity chromatography

Uses of PCR and RTPCR

• detection of DNA/RNA fragments (for

diagnostic purposes)

• amplification of DNA for detection of

polymorphism and other analytical
techniques (sequencing, DGGE [and related
techniques], etc.)

• cloning of genes (degenerate primers,

differential display)

• mutation of genes

• gene construction
 PCR-based
detection of
polymorphism

Comparing genomes:
The Random Amplified Polymorphic DNA
(RAPD) technique
 Comparing genomes:
The Amplified Fragment Length Polymorphism
(AFLP) technique

Comparing genomes:
The Restriction Fragment Length Polymorphism
(RFLP) technique
Detection of gene polymorphism

PCR-RFLP
 Temperature Gradient Gel Electrophoresis
(TGGE)

increasing
temperature

Variation:
• Temporal Temperature Gradient Gel Electrophoresis
• Denaturing Gradient Gel Electrophoresis

(non-denaturing gel)

Exonuclease may be used to digest one of the two strands;

primer must be phosphorylated
 Differential Display-RTPCR (DDRT-PCR)

a technique for identifying

differentially expressed genes

Differential Display-RTPCR: principle

tissue1 tissue2 tissue3 tissue(n)

extract mRNA

RT-PCR

polyacrylamide-gel
electrophoresis
Differential Display-RTPCR:
example

ddRT-PCR of mycelial and

budding Candida albicans

Pulsed-field gel electrophoresis

standard agarose gel electrophoresis

 Pulsed-field gel electrophoresis

orthogonal field alternation gel electrophoresis

DNA labeling

• synthesis of DNA probes

• detection of specific fragments

 DNA labeling
• Types of labels
• radioisotopes (e.g., [α-32P] dNTP)
• fluorescent groups (fluorophores)
• enzymes
• fluorescent substrates
• chromogenic substrates
• labeling strategies
• 5’ or 3’ labeling of fragments ( using T4 kinase or
deoxynucleotidyl terminal transferase)
• incorporation of labeled nucleotides during DNA
synthesis
• covalent attachment of enzymes to DNA fragments

Labeled nucleotides: examples

32P
DNA labeling with dyes or haptens:
example of a two-step strategy