PCRPCRA technique to make a A technique to make a lot of DNA from only a lot of DNA from only a

little!little!

PCRPCR

►Full name of the process is Polymerase Full name of the process is Polymerase Chain ReactionChain Reaction

► Invented in 1983 by Dr. Kary MullisInvented in 1983 by Dr. Kary Mullis►Allows scientists to make unlimited Allows scientists to make unlimited

copies of specific DNA fragmentscopies of specific DNA fragments►Can amplify DNA fragments of up to Can amplify DNA fragments of up to

approximately 10,000 bpapproximately 10,000 bp

PCRPCR

Two problems exist for scientists to Two problems exist for scientists to make a visual analysis of a specific make a visual analysis of a specific DNA locus (site or location):DNA locus (site or location):

1.1.Must first Must first isolateisolate the specific the specific locus from all the other millions locus from all the other millions of sites in chromosomesof sites in chromosomes

2.2.Must Must amplifyamplify (duplicate) the (duplicate) the locus so that you can see it on locus so that you can see it on the gelthe gel

PCR RequirementsPCR Requirements

1.1. Template DNA (what you extract)Template DNA (what you extract)

2.2. PrimersPrimers

3.3. DNA polymeraseDNA polymerase

4.4. DNTP (deoxyribonucleoside DNTP (deoxyribonucleoside triphosphates) triphosphates) oror the A’s, T’s, C’s, the A’s, T’s, C’s, and G’sand G’s

5.5. Thermal CyclerThermal Cycler

PCR RequirementsPCR RequirementsPrimersPrimers► Short segments of DNA 20-30 bp Short segments of DNA 20-30 bp

long which “bracket” the desired long which “bracket” the desired DNA segmentDNA segment

► One primer is a complement One primer is a complement “forward” primer to produce DNA “forward” primer to produce DNA strand from left to right while one is strand from left to right while one is a “reverse” primer that is for right a “reverse” primer that is for right to left strandto left strandA T T C G C G A A A T G T T G G G C A A C A G G T A C

T C T A

T A A G C G C T T T A C A A C C C G T T G T C C A T G A G A T

C A G G T A C T C

G C G C T T T A C

PCR RequirementsPCR Requirements

► If primers are carefully chosen they If primers are carefully chosen they can select a unique sequence in the can select a unique sequence in the genomegenome

PCR RequirementsPCR RequirementsHeat stable DNA polymeraseHeat stable DNA polymerase► Most commonly use Most commonly use Taq Taq

polymerasepolymerase – – ► Thermus aquaticusThermus aquaticus (a bacteria (a bacteria

found around hot springs)found around hot springs)► Is an enzyme that helps form Is an enzyme that helps form

new bonds between the new bonds between the nucleotides in new strands of nucleotides in new strands of bacteriabacteria

A T T C G C G A A A T G T T G G G C A A C A G G T A C T C T A

T A A G C G C T T T A C A A C C C G T T G T C C A T G A G A T

C A G G T A C T C

G C G C T T T A C

PCR RequirementsPCR RequirementsDNTP’sDNTP’s► DDeoxyriboeoxyribonnucleoside ucleoside

ttriripphosphates – hosphates – ► Nitrogen bases: adenine, Nitrogen bases: adenine,

thymine, cytosine, and guaninethymine, cytosine, and guanine► These DNTP’s attach to the These DNTP’s attach to the

exposed complementary bases exposed complementary bases of the original DNAof the original DNAA T T C G C G A A A T G T T G G G C A A C A G G T A C

T C T A

T A A G C G C T T T A C A A C C C G T T G T C C A T G A G A T

C A G G T A C T C

G C G C T T T A C

A A C C C G T T G T C C A T G A G A T

A T T C G C G A A A T G T T G G G C A A

PCR RequirementsPCR Requirements

Ready to go Ready to go PCR BeadsPCR Beads► For convenience, we use PCR beads For convenience, we use PCR beads

which contain freeze-dried DNTP’s which contain freeze-dried DNTP’s and and TaqTaq polymerase enzyme polymerase enzyme

► All we add to them are the primer All we add to them are the primer mix and template DNAmix and template DNA

**Note****Note**These beads are These beads are veryvery expensive expensive – –

Almost $2.00 per bead!!! So Almost $2.00 per bead!!! So don’t mess it up!!don’t mess it up!!

PCR: The Steps of the PCR: The Steps of the ProcessProcess

►3 phases for each cycle (these vary 3 phases for each cycle (these vary slightly from one protocol to another)slightly from one protocol to another) DenaturingDenaturing: (94-95°C) DNA strands : (94-95°C) DNA strands

separate into single strandsseparate into single strands AnnealingAnnealing: (58°C) Primers anneal : (58°C) Primers anneal

(attach) to the separated DNA strands(attach) to the separated DNA strands ExtendingExtending: (72°C) New : (72°C) New

complementary strands are made as complementary strands are made as the the TaqTaq enzyme helps to form bonds enzyme helps to form bonds with the DNTP’s with the DNTP’s

PCR: The Steps of the PCR: The Steps of the ProcessProcess

►Approximately Approximately 30 cycles 30 cycles of these 3 of these 3 phases are usedphases are used

►Each cycle produces twice as many Each cycle produces twice as many targeted DNA segments as existed targeted DNA segments as existed beforebefore

►After 30 cycles approximately 1 After 30 cycles approximately 1 billionbillion copies are produced copies are produced

►Takes approximately 2 hoursTakes approximately 2 hours

PCR Equipment: Thermal PCR Equipment: Thermal CyclerCycler

PCR Equipment: Thermal PCR Equipment: Thermal CyclerCycler

► It is possible to substitute 3 It is possible to substitute 3 water water baths baths at the 3 different at the 3 different temperatures for a thermal cycler, temperatures for a thermal cycler, but…but…

►DNA samples would have to be DNA samples would have to be moved from water bath to water moved from water bath to water bath to water bath for 30 cycles!bath to water bath for 30 cycles!

PCR Activity: Paper PCRPCR Activity: Paper PCR

►Use strips of paper to create Use strips of paper to create 4 cycles 4 cycles of of a PCR reaction.a PCR reaction.

►Color code the reactions as follows:Color code the reactions as follows:RedRed: Template DNA strand (40 : Template DNA strand (40 bp)bp)Write the following sequence on Write the following sequence on one red strip:one red strip:

A C G A C G A T TA T T G G G G G G C C AC C A A T A A T A T A CT A C G C G C G G T G AT G A T G T T G T T C GT C G A A G A A G A G AA G A C T C T

PCR Activity: Paper PCRPCR Activity: Paper PCR

YellowYellow: 1: 1stst copied strand copied strand BlueBlue: 2: 2ndnd copied strands copied strands GreenGreen: 3: 3rdrd copied strands copied strands

Forward primersForward primers are are

whitewhite Reverse primers are Reverse primers are

graygray G A T T C C A T A

Steps of DNA Isolation:Steps of DNA Isolation:

►10 mL of saline, swish, spit, use 1mL of 10 mL of saline, swish, spit, use 1mL of this.this.

►CentrifugedCentrifuged►Poured off saline & then resuspendedPoured off saline & then resuspended►Removed 30 uL and added to 100 uL of Removed 30 uL and added to 100 uL of

Chelex to absorb cell debris and ions.Chelex to absorb cell debris and ions.►Boiled for 10 minutes.Boiled for 10 minutes.►Withdrew 30 uL & stored in new tube.Withdrew 30 uL & stored in new tube.

Day 2: AmplificationDay 2: Amplification

►Added 22.5 uL of Primer Mix into PCR Added 22.5 uL of Primer Mix into PCR bead.bead.

►Added 2.5 uL of DNA.Added 2.5 uL of DNA.►Quick spin to put everything together.Quick spin to put everything together.►Thermal cyclerThermal cycler

►DAY 3: Ran gelsDAY 3: Ran gels