Polymerase Chain reaction (ADD VIDEO STUFF)

PCR

Polymerase Chain Reaction (PCR) is a molecular Genetics technique to amplify and isolate specific sequences of DNA.

DNA Polymerase is an enzyme that That is most active around 72°C.

Chain reaction refers to making large numbers of copies of the DNA sequence.
Think of PCR as a biotechnology that is like a DNA photocopier.

Materials needed for DNA replication:

DNA polymerase

•  solutions containing nucleotides (dATP, dCTP, dGTP, dTTP)

• DNA template,

• DNA primers,

• pH buffer

• ions (eg. Mg2+) 

There are three repeating steps: 

denaturation

• annealing

• extension

Steps for PCR in detail

1. The mixture is heated to 95°C. The hydrogen bonds between the strands of the template double helix DNA molecules denature to leave two single-stranded DNA molecules.

2. Then the mixture is cooled to 45-65°C.   Double stranded helices form between complementary DNA molecules, including the annealing of primers to the template.

3. The mixture is heated and the new DNA strand is synthesized. 

idk what step extension at 72 degrees

What does pcr stand for and why is that the name

polymerase chain reaction

DNA Polymerase is an enzyme that is most active around 72°C.

Chain reaction refers to making large numbers of copies of the DNA sequence. 

A Primer is a short sequences of about 20 nucleotides that provides a starting point for DNA synthesis.   Primers bind,  “inward” in the 5’ to 3’ direction towards the region to be copied.

what is it analagous to

a stereo amplifier photocopies multiple copies if a certain segment of DNA

analogous to a radio amplifier. Radiowave signals, not normally not heard, are amplified so we can hear music.

– small amounts of DNA are amplified to allow analysis

Applications

first was developing sickle cell anemia. used for genome projects for DNA mapping and sequencing

in all these cases DNA samples that are extracted are limited and PCR amplifies segments of DNA that become the subject of further analysis and study

Steps

In a PCR reaction, the first step is the preparation of the DNA sample (denature DNA) that is extracted from tissues or various biological sources. A set of two primers (a forward and reverse primer) usually ranging between 20 and 45 nucleotides are chemically synthesized to correspond to the two ends of the gene to be amplified. Each primer binds to one of the two chemically synthesized to correspond to the two ends of the gene to be amplified. Each primer binds to one of the two DNA strands and is the initiation point of the amplification. The primer concentrations are always in excess of the target gene to make possible subsequent priming. The exact nucleotide primer sequences for a specific amplification reaction are determined to yield the best conditions (hybridization) for template-primer formation

A typical PCR mixture

contains DNA, Taq DNA polymerase, and the four deoxynucleotide triphosphates in the appropriate buffer

incubation mixture is then exposed to a three step

The first temperature is 94 degrees to melt the H bonds between DNA strands. The temp is then dropped to between 42 and 60 degrees to hybridize the primers on the two target DNA strands.The temp is then increased to 72 degrees which is optimum temp for taq DNA polymerase. At this temperature, the DNA polymerase synthesizes the opposite strand of DNA using original strands as templates. These temperature cycles are repeated 20-50 times.

how is this process made efficient

This process is made efficient by placing the reaction tubes in specifically designed thermal cycles which are programmed to alternate temperatures rapidly or accurately. The amplified product is then detected by separating the reaction mixture by gel electrophoresis and analysis

look at the multiple choice stuff

!!

• who first described the procedure in 1985

• identify, define ,and explain the components used in a PCR reaction

• explain the three phases in one cycle of PCR and their approximate temperatures

GET THIS FROM FIRST VIDEO

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Materials needed for DNA replication:

DNA template segment to copy (pattern make new strands of DNA)

• DNA polymerase enzyme - create DNA strands where primers indicate- often Taq enzyme from Thermus aquaticus, a heat-tolerant bacterium of hotsprings 

• 4 different types of DNA nucleotides (dATP, dCTP, dGTP, dTTP)

• DNA primers -tells where to start building

• Salt environment: pH buffer and ions (eg. Mg2+) 

There are three repeating steps: 

denaturation   (separates the complementary strands of DNA

• annealing        (identifies the section of DNA to be copied)

• extension        (copies the DNA)

What is the PCR process?

1. The mixture is heated to 95°C. The hydrogen bonds between the complementary strands of the template double helix DNA molecules denature (breaks) to two single-stranded DNA molecules (Heat is used to denature (separate) the strands).

2. Then the mixture is cooled to 45-65°C.   The annealing (attach/binding) of primers to each target DNA strand to be amplified (copied).  

3. The mixture is heated and then extend the new DNA strands areas where the primer and template have annealed.  Polymerase (P) finds the ends of short double stranded regions of the DNA where the primers were bound.  Polymerase ( e.g. Taq) adds the correct complementary DNA nucleotides together to make new strands.

who first described the procedure

Kary Mullis in 1985

Seven steps of PCR experiment

Bench of two groups will use a strip of 8 PCR tubes: +wh +wh control

1. label tubes

2. Add to each tube: PCR EdvoBead, (yellow) 20 microlitres Primer Mix, (red) 5 microliters of extracted DNA. EDVObead contains dNTP’s, DNA taq polymerase, DNA taq polymerase buffer MgCl2

3. gently mix= flick dissolve in each tube completely; mizture now is orange

4. Instructor will make one control per two groups

5. spin: centrifuge to collect samples at bottom of tube. Empties at opposite ends.

6. Thermocycler: Amplify DNA using PCR. Wait until 4 degrees then take out

7. push down on cap to resnap before putting in freezer. Store at -20 C for future separation of products for electrophoresis

Components in a PCR reaction:

• Template (the DNA you want to amplify) • 2 different Primers: Sequence-specific DNA molecules at ends of the target sequence: • 4 Nucleotides (dATP, dCTP, dGTP, dTTP)- building blocks of DNA • Thermal Cycler- PCR machine rapidly heats and cools samples • Magnesium ions (enzyme cofactor) • Buffer, containing salt • Taq DNA polymerase- the heat-resistance enzyme extracted from bacteria from hotspringsThermus aquaticus

Three steps in the PCR cycle

Denaturation – heated to 94oC unzips the strands (breaking of H bonds between strands)

Annealing – primers anneal to target DNA sequence (• Primers (forward and reverse) bind to the template sequence. Each primer is the initiation point of amplification. • Taq polymerase binds to double-stranded substrate) primers anneal at about 55C

Extension/Elongation – at 72oC Taq polymerase extends the primer to synthesize a new strand of DNA. (Taq polymerase extends primer; synthesizes the opposite strand of DNA using the original strand as a template • DNA is replicated Extends at 72°C; optimal temperature for Taq DNA polymerase)

importance of many cycles

30 cycles allow production of enough DNA for analysis'

LOOK AT DIAGRAM ON SLIDE 13 be able to label