• PCR way of copying specific DNA fragments from small sample DNA material "molecular photocopying"

• It’s fast, inexpensive and simple

Polymerase Chain Reaction

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)

• The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA

• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

The three main steps of PCRStep 1: Denature DNA At 95C, the DNA is denatured (i.e. the two strands are

separated)

Step 2: Primers Anneal At 40C- 65C, the primers anneal (or bind to) their

complementary sequences on the single strands of DNA

Step 3: DNA polymerase Extends the DNA chain At 72C, DNA Polymerase extends the DNA chain by adding

nucleotides to the 3’ ends of the primers.

PCR: Polymerase Chain Reaction

Step 1: denaturation

Step 2: annealing

Step 3: extension

DNA Replication vs. PCR

PCR is a laboratory version of DNA Replication in cells

in vitro in vivo

DNA Replication enzymes:

• DNA Polymerase- builds DNA strand

• DNA Ligase- joins DNA strand together

• Primase- short RNA sequence that serves as a starting point for DNA synthesis

DNA Replication enzymes:• Helicase untwists the two parallel DNA strands

• Topoisomerase relieves the stress of this twisting

• Single-strand binding protein binds to and stabilizes the unpaired DNA strands

DNA Synthesis

PCR: the in vitro version of DNA Replication

The following components are needed to perform PCR in the laboratory:

1) DNA (your DNA of interest that contains the target sequence you wish to copy)

2) A heat-stable DNA Polymerase (like Taq Polymerase)3) All four nucleotide triphosphates4) Buffers5) Two short, single-stranded DNA molecules that serve as

primers6) Thin walled tubes7) Thermal cycler (a device that can change temperatures

dramatically in a very short period of time)

PCR

PCR C1000 Thermal Cycler

PCR tubes

Denaturation of DNA

This occurs at 95 ºC mimicking the function of helicase in the cell.

Step 2 Annealing or Primers Binding

Primers bind to the complimentary sequence on the target DNA. Primers are chosen such that one is complimentary to the one strand at one end of the target sequence and that the other is complimentary to the other strand at the other end of the target sequence.

Forward Primer

Reverse Primer

Step 3 Extension or Primer Extension

DNA polymerase catalyzes the extension of the strand in the 5-3 direction, starting at the primers, attaching the appropriate nucleotide (A-T, C-G)

extension

extension

• The next cycle will begin by denaturing the new DNA strands formed in the previous cycle

The Size of the DNA Fragment Produced in PCR is Dependent on the Primers

• The PCR reaction will amplify the DNA section between the two primers.• If the DNA sequence is known, primers can be developed to amplify any

piece of an organism’s DNA.

Forward primer

Reverse primer

Size of fragment that is amplified

The DNA of interest is amplified by a power of 2 for each PCR cycle

• For example, if you subject your DNA of interest to 5 cycles of PCR, you will end up with 25 (or 64) copies of DNA. (25x 2 strands= 64)

• Similarly, if you subject your DNA of interest to 35

cycles of PCR, you will end up with 235 (or 68,719,476,736) copies of DNA!

I. Polymerase Chain ReactionThe very first thing you will do when you arrive for the lab activity will be to set up the PCR reactions and run them in the thermal cycler.

Be sure to keep all of your reagents cold (on ice) throughout the procedure.

Each group (a pair of students) will be given a tube containing a “PCR master mix” containing all of the necessary reagents including Taq polymerase, except for your DNA template (your DNA extracted from the bacteria in the previous lab activity).

Procedures and Assignment

• PCR master mix with Taq polymerase is all ready prepared

• Provided are the following amounts for each reaction:

40.5 μL sterile water5 μL 10X buffer1.5 μL MgCl20.5 μL dNTP’s0.5 μL of each F8 primer0.5 μL of each R515 primer0.5 μL Taq (added last)

PCR master mix

dNTPsF8 & R515 Primers

Buffer

+ +water

MgCl2

Taq

Keep on ice

1 μL DNA

49 μL

PCR Tube

Thermocycling Program

• Files (F2)• Users (enter)• PACES

• 165RRNA• RUN

Program Thermal Cycler

Denature: 95oC for 4 min

Then 35 cycles:Denature: 95oC for 30 secAnneal: 55oC for 30 secExtend: 72oC for 30 sec

Final Extension: 72oC for 7 minutesFinal Hold: 4oC

Takes 1 hr 44 min

Thermocycling protocol

annealing

95ºC 95ºC

55ºC

72ºC

4ºC

4 min 30 sec

30 sec

30 sec

∞ hold

Initial denaturation of DNA

1X 35X 1X

extension

denaturation

Final extension

72ºC

7 min

• After completing the PCR, place PCR tube in holder and back in freezer

• It will be used for electrophoresis next week.

II. PCR DNA