Lecture 2Mandatory Articles/Finding structure on the web.

Restriction enzymes:

a) History

b) uses/properties

PCR:

a) basics

b) considerations

Agarose Gel electrophoresis

Tips for this week’s lab

Mandatory Articles• Sept. 29 Article 1• Oct. 6 Article 2• Oct. 13 Thanksgiving • Oct. 20 Article 3• Oct. 27 Article 4• Nov. 3 Article 5• Nov. 10 Article 6• Nov. 17 Article 7

Finding structures on the Web

• http://www.ncbi.nlm.nih.gov/

• Choose structures

• Search for the one you are interested in.

• Download file and viewer and look at your structure.

Socratic Method• Questions are the fundamental guiding

element that lead to answers, or boundaries to the unanswerable.

• Method of critical thinking/investigation.

• Development of student intellectual autonomy.

• I use a loose Socratic style in lecture.

Restriction enzymes• Enzymes and recombinant DNA

technology.

• How were restriction enzymes found originally?

• What are the properties of restriction enzymes?

• What are their uses?

Early 1970s: Genomic Studies: How to cut DNA into manageable fragments? Chemical/mechanical means non-specific, non-reproducible

Breakthrough needed

Restriction Endonucleases: Molecular Scissors for Cutting DNA

But where did restriction enzymes come from?

• They were discovered as a method of protecting bacteria from bacteriophage infection.

• Called the restriction modification system

Restriction Enzymes Recognize Palindromic Sequences

Restriction Enzyme Recognition site

EcoRI 5’ GAATTC 3’

HindIII 5’ AAGCTT 3’

BamHI 5’ GGATCC 3’

Restriction enzymes

Considerations

Why do we have MgCl2 ?

Why do we have NaCl?

Why do we have a buffer?

Restriction enzymes can be used to

create a “MAP” of DNA

? MAP ?

Cleavage of DNA with restriction enzymes provides landmarks and sequence information.

Restriction enzymes can be used to create a “MAP” of DNA

Digestion of DNA with restriction enzymes cloning into vectors

Construction of restriction mapsof individual clones

Restriction enzymes played a pivotal role in cloning of the Human Genome

FORMATION OF RECOMBINANT DNA

Polymerase Chain ReactionConsiderations

Why do we have MgCl2?

Why do we have a buffer?

Why do we have dNTPs?

Why do we have primers?

Why three temperatures per cycle?

POLYMERASE CHAIN REACTIONSTEPS INVOLVED

Taq Polymerase

Forward PrimerReverse Primer

Unlike most enzymes, Taq DNA polymerase can withstand high temperatures necessary for DNA strand separation and can be left in the reaction.

PCR: Representative Temperature Profile

AMPLIFICATION OF DNA

POLYMERASE CHAIN REACTION

POLYMERASE CHAIN REACTION (Con’t)

EXPONENTIAL AMPLIFICATION OF PRODUCT

1.  Primers should be 18-25 bases in length;   2.  Base composition should be 50-60% (G+C);   3.  Primers should end (3') in CG or GC: this creates “tight” ends and increases efficiency of priming   4.  Tm = 4(G + C) + 2(A + T) oC: Ta should be 2-5 oC below Tm( 55-60oC) 5.  3'-ends of primers should not be complementary: primer-dimer6.  Primer self-complementary (ability to form hairpins) should be avoided.  7.  Three or more Cs or Gs at the 3'-ends of may promote mispriming at G or C-

rich sequences (because of stability of annealing), and should be avoided. 

DESIGNING PRIMERS

Adapted from Innis and Gelfand, 1991

Gel electrophoresisConsiderations

On what basis are molecules separated?

How does a gel work?

Which way do molecules go and why?

What colour are electrodes and anodes?

AGAROSE GEL ELECTROPHORESIS

Agarose: A polysaccharide extracted from seaweed. Used to separate DNA fragments based on size

Structure of Ethidium Bromide (Fluorescent Dye)

Detecting DNA using Ethidium Bromide

When excited by UV light, EtBr emits fluorescent light at 590 nm

EtBr is a very dangerous mutagen.

Intercalates between bases of DNA

How are different sizes of DNA strands separated on agarose gel?

Mixture of DNA molecules

DNA separation is based on fragment size

Gel of the week

Decreasing S

ize

(+)

(-)

PCR1PCR2

Size markers and their use to determine size

Size markers and their use to determine size

Mobility of a DNA fragment is proportional to the log of itssize in bases.

Plot of relative mobility of DNA vs log of size on semi-log graph paper

Varying concentrations of agarose

How does one make a 0.7%, 1.0% & 1.5%agarose gel?

Why are there varying concentrationsof agarose gels?

DNA Ladders

Varying concentrations of agarose

Why are there varying concentrationsof agarose gels?

Higher concentrations provide better resolution for smaller DNA fragments

Lower concentrations provide better resolution for larger DNA fragments

DNA Ladders

Tips• Major reason for this experiment not

working is not having all the reaction components in the correct tube.

• Tick list

• Droplets on tube wall

• Master Mix

Tick listComponent Tube 1 Tube 2

10Xbuffer4μl

X X

MgCl21μl

X

Enzyme0.5μlWater13μl

Droplet method

Droplet method

• Check pipet tip for solution.

• Eject solution on the side of tube.

• Check for a droplet.

• To get the solution in the droplet to the bottom of the tube tap the tube on the bench.

Master Mix

On the board.

Gel of the week

100bp Ladder .1 .5 1 2 NTC .1 .5 1 2

.1 .5 1 2 .1 .5 1 2

.1 .5 1 2 .1 .5 1 2

DNA Buffer MgCl2 Primer 1 dNTPs Taq

Concentration (X)

100bp Cycle #

ladder 5 10 15 20 25 30