amp 9 Polymerase Chain Reaction PCR General Genetics Objectives 1Introduce the students to the preparation of the PCR reaction 2Examine the PCR products on agarose gel electrophoresis ID: 369160
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Slide1
Lab # 8 & 9Polymerase Chain Reaction (PCR)
General GeneticsSlide2
Objectives:1.Introduce the students to the preparation of the PCR reaction.
2.Examine the PCR products on agarose
gel electrophoresis.
3.Explain some of
PCR
applications.Slide3
Introduction:Kary mullis
received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR).
The process, which
Kary
Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. Slide4
PCR :PCR is used to amplify specific regions of a DNA strand (the DNA target) in vitro. This can be a single gene
, a part of a gene, or a non-coding sequence.
Most PCR methods typically amplify DNA fragments of up to
10
kilo base pairs
(kb), although some techniques allow for amplification of fragments up to
40 kb
in size. Slide5Slide6Slide7
Components of PCR reaction:The solution must include:The template DNA A thermostable
DNA polymerase Two oligonucleotide primers
Deoxynucleotide
triphosphates
(
dNTPs
)
Reaction buffer (
Tris
, ammonium ions (and/or potassium ions), magnesium ions, bovine serum albumin).
This components must be mixed together in a solution with
a total volume
of between 25 and 100
microliters
.Slide8
Cont.Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (
amplicon) in the reaction. At 30 cycles there are 1,073,741,764 target copies (~1×109).Slide9Slide10Slide11Slide12Slide13
PCR procedures : stepsEach cycle of PCR includes steps for DNA template :1. Denaturation : 94°C 15 sec_2 min 2. Primer annealing: 40–60°C 15 sec_60 sec
3. Primer extension: 70–74°C 1–2 minutesSlide14
First step: Denaturation :Denatures the target DNA by heating it to 94°C for 15 seconds to 2 minutes. The two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase.Slide15
Denaturation
HeatingSlide16
second step: primer annealing:The temperature is reduced to approximately 40–60°C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds.Slide17
primer annealingSlide18
Third step: primer extensionThe synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70–74°C.
The extension step lasts approximately 1–2 minutes. Slide19
ExtensionSlide20
Cont.The next cycle begins with a return to 94°C for denaturation. After 30–40 cycles, the amplified product may then be analyzed for size, quantity, sequence, etc., or used in further experimental procedures.Slide21Slide22Slide23
Thermus aquaticus :Is a species of bacterium that can tolerate high temperatures one of several thermophilic bacteria. It is the source of the heat-resistant enzyme
Taq DNA Polymerase, one of the most important enzymes in molecular biology because of its use in the PCR (DNA amplification technique). Taq produces an enzyme called DNA polymerase, that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg.Slide24Slide25Slide26