Presentation on theme: "PCR way of copying specific DNA fragments from small sample DNA material "molecular photocopying" It’s fast, inexpensive and simple Polymerase Chain Reaction."— Presentation transcript:
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 PCR Step 1: Denature DNA At 95 C, the DNA is denatured (i.e. the two strands are separated) Step 2: Primers Anneal At 40 C- 65 C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA Step 3: DNA polymerase Extends the DNA chain At 72 C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.
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
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 triphosphates 4)Buffers 5)Two short, single-stranded DNA molecules that serve as primers 6)Thin walled tubes 7)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
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 2 5 (or 64) copies of DNA. (2 5 x 2 strands= 64) Similarly, if you subject your DNA of interest to 35 cycles of PCR, you will end up with 2 35 (or 68,719,476,736) copies of DNA!
I. Polymerase Chain Reaction The 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 μ L sterile water 5 μ L 10X buffer 1.5 μ L MgCl μ L dNTP’s 0.5 μ L of each F8 primer 0.5 μ L of each R515 primer 0.5 μ L Taq (added last) PCR master mix dNTPs F8 & R515 Primers Buffer + water MgCl 2 Taq Keep on ice 1 μL DNA 49 μL PCR Tube
Thermocycling Program Files (F2) Users (enter) PACES 165RRNA RUN
Program Thermal Cycler Denature: 95 o C for 4 min Then 35 cycles: Denature: 95 o C for 30 sec Anneal: 55 o C for 30 sec Extend: 72 o C for 30 sec Final Extension: 72 o C for 7 minutes Final Hold: 4 o C Takes 1 hr 44 min
Thermocycling protocol annealing 95ºC 55ºC 72ºC 4ºC 4 min 30 sec ∞ hold Initial denaturation of DNA 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