lecture 2 mandatory articles/finding structure on the web. restriction enzymes: a) history b)...
TRANSCRIPT
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