Molecular Biology Lab Techniques: Hybridization, Restriction Enzymes, PCR, Lab Reports of Biology

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Basic lab techniques

Sandrine Dudoit

Bioconductor short course

Summer 2002

© Copyright 2002, all rights reserved

Lab techniques

Why?

• Why cut, amplify, sort, probe?

• Genotyping (cf. genetic mapping,– Sequencing;

forensics);

• Etc.– Measuring gene expression;

Hybridization

Hybridization

refers to the

annealing

base-pairing rules. of two nucleic acid strands following the

• Nucleic acid strands in a duplex can be

separated, or

denatured

, by heating to

destroy the hydrogen bonds.

Restriction enzymes

DNA

restriction enzymes

or

restriction

endonucleases

recognize short, specific sequences

phosphate backbone of the DNA.of DNA bases and make breaks in the sugar-

The recognition sites are usually

palindromes

, .i.e,

other strand, read in the reverse direction.the sequence in one strand is the same as that in the

stranded ends orthe opposite strand, creating complementary, single-Some restriction enzymes make staggered cuts in

sticky ends

; others cut across both

strands creating DNA fragments with

blunt ends

EcoRI

• Restriction enzymes allow bacteria to

containing organisms (e.g. virus).self-defend against invading DNA-

• EcoRI, from

Escherichia coli

or

E. coli.

5' G

AATTC

3' CTTAA

G

Restriction enzymes

http://www.ultranet.com/~jkimball/BiologyPages/

Restriction enzymes

PCR

PCR relies on

• Known sequence for the 3' end of the

template

i.e., segment to be amplified.

• Availability of

primers

, i.e., synthetic

the template.oligonucleotides complementary to the 3' ends of

• Use of

temperature

to control DNA

annealing

and

denaturation

• Existence of a temperature resistant enzyme for

DNA synthesis by primer extension:

Taq

polymerase

Thermus aquaticus

, bacterium found

in Yellowstone hot springs).

PCR

Main ingredients:

DNA template,

primers in great excess of template,

dNTPs: deoxynucleotide triphosphates,

Taq polymerase.

Repeated

cycles

of DNA denaturation

provide many copies of the template.(heating) and synthesis (cooling) rapidly

which are repeated for 30 or 40 cycles.There are three major steps in a PCR,

PCR

PCR

PCR

Taq polymerase

http://berget.mcs.cmu.edu/education/TechTeach/replication/TaqI.html