survey of molecular techniques

8/9/2019 Survey of Molecular Techniques

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Survey ofmolecular techniques

Enzymes for DNA manipulation

DNA polymerases

nucleases

ligases

end-modification enzymes


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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


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Exonucleases: examples

Exonuclease III - removes residues from 3 ends

of a DNA strand

Bacteriophage exonuclease - removes residues

from 5 ends of a DNA strand

Endonucleases

cut at internal phosphodiester bonds

restriction enzymes - an important class ofDNA endonucleases; play a central role in

recombinant DNA technology


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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 thebacteria it was isolated from

For example:EcoRI (pronounced eeko-are-one)

E = Genus, Escherichia

co = Species, coli

R = Strain, RY13

I = First endonuclease isolated fromthis bacteria


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Restriction Sites

Specific sequence of nucleotides that aparticular restriction enzyme recognizes

Most restriction sites are about 4-6 baseslong

Many are palindromic

- GGATCC -

- CCTAGG -

Frequency that a restriction site is likely tooccur in a strand of DNA can be predicted

1/4n where n = # of bases in the restriction siteA four base pair restriction site will occur every

256 bases

Types of ends after restriction


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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


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5

3

G

C T T A A

A A T T C

G

one DNA fragment another DNA fragment

Ligation of DNA fragments

annealing of sticky ends

G A A T T C

C T T A A G3

5 3

5

G A A T T C

C T T A A G

DNA ligase-catalyzed ligation

+

Ligation of DNA fragments

Ligation of joining of double strands vs.

ligation of nicked fragments


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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

++


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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 phosphataseremoves phosphate group from 5 ends

Polymerase Chain Reaction

(PCR)


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Polymerase Chain Reaction (PCR)

94 oC

1 min 50 oC

20 s

72 oC

1 min

denaturation

annealing

of primers

extension

DNA

Parameters of a PCR cycle:an example

n

oC


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Thermal cycler:

The PCR machine

Agarose gel electrophoresisGel: agarose, 0.7 - 2.5 %

+

_


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Ethidium bromide

as a fluorescent stain

Agarose gel electrophoresis: analysis

Detection of DNA

fragment

determining sizes of

fragments

size

marker


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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)


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Variations of PCR

nested PCR

inverse PCR

panhandle PCR

quantitative PCR

multiplex PCR

asymmetric PCR

in situPCR etc.

cDNA


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Isolation of mRNA by affinity chromatography

detection of DNA/RNA fragments (fordiagnostic purposes)

amplification of DNA for detection ofpolymorphism and other analyticaltechniques (sequencing, DGGE [and related

techniques], etc.)

cloning of genes (degenerate primers,differential display)

mutation of genes

gene construction

Uses of PCR and RTPCR


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PCR-based

detection of

polymorphism

Comparing genomes:The Random Amplified Polymorphic DNA

(RAPD) technique


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Comparing genomes:

The Amplified Fragment Length Polymorphism

(AFLP) technique

Comparing genomes:

The Restriction Fragment Length Polymorphism(RFLP) technique


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Detection of gene polymorphism

PCR-RFLP


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Temperature Gradient Gel Electrophoresis

(TGGE)

increasing

temperature

Variation:

Temporal Temperature Gradient Gel Electrophoresis Denaturing Gradient Gel Electrophoresis

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

primer must be phosphorylated

(non-denaturing gel)


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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-gelelectrophoresis


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Differential Display-RTPCR:

example

ddRT-PCR of mycelial and

budding Candida albicans

Pulsed-field gel electrophoresis

standard agarose gel electrophoresis


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Pulsed-field gel electrophoresis

orthogonal field alternation gel electrophoresis

DNA labeling

synthesis of DNA probes

detection of specific fragments


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DNA labeling

Types of labels

radioisotopes (e.g., [-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


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

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