1. Polymerase Chain Reaction (PCR)
Biotechniques (BIOL 410)
Polymerase Chain Reaction (PCR)

2. DNA Replication
Cells poses a natural process for creating new strands of DNA
Base-pairing
Nucleotides will form an aligning bond with a specific complementary base
Therefore, the two strands are complementary
Each can be used as a template for a new strand

3. DNA Replication Original strand of DNA unwound
The two complementary strand separate
Each is used to create a new complementary strand
DNA has been duplicated

4. Complementing Bases Adenine and Thymine form two h-bonds
Guanine and Cytosine form three h-bonds
Prevents miss-matching
Allows one strand to act as template and form a complementary strand

5. Complementary Base Pairing

6. DNA Polymerase Polymer = strand
-ase = Enzyme
Attaches to Primer and moves along the template (DNA)
Add new nucleotides to the new complementary chain
Sliding clamp moves behind the DNA Polymerase, causing the newly added DNA nucleotides to properly attached to the complementing bases and the sugar phosphate backbone
Part of the DNA Polymerase complex

7. Initiation of DNA Replication
Several Protein Structures involved in replication
Move along the DNA like a train on tracks

8. Polymerase Chain Reaction
In vitro form of DNA replication
Instead of copying entire genome, will only copy a specfic segment of DNA

9. PCR
Using PCR a single strand of target DNA can be used to create BILLIONS of copies
In hours
In vitro

10. History
Concept first published in 1971 by Kjell Kleppe & Godbind Khorana (Norway)
Published their concept of using enzymatic reactions to copy short regions of DNA
Kerry Mullis (Cetus Corp.)
Designed a methods for synthesizing short regions of DNA in vitro
1993 Nobel Prize in Chemistry (Biochemistry)
Khorana
Khorana – 1968 Nobel Laureate in Biochemistry for demonstrating Codon sequencing

11. Controversy in the Ivory Tower
Cetus’ owned the patents for PCR, since Mullis developed the method while in their employment
One of the first Biotech companies
Patent was purchased by La Roche Pharmaceuticals
But the patents for the method and Taq (the enzyme) have been challenged for years by companies such as DuPont and Promega

12. PCR Process Three-step process
Denaturation – the two strands of DNA are seperated
Anneling – The primers are adhered to the proper complementry sequence on the single DNA strands
Elongation – Taq binds to the primers and begins forming the complementary strand
Thermocycler – Use temprature to control the reactions

15. 1986 Prototype PCR machine (Thermocycler)
1986 Prototype PCR machine (Thermocycler). This machine was called “Baby Blue”

16. Thermocycler Thermocyclers will control the temprature
based on a program that includes denaturing, annealing, and elongation conditions
Repeats the cycle, doubling the DNA product with each cycle

17. Master Mix
Master Mix – a cocktail of ingriedents necessary for DNA Polymerase
Primers – short single-stranded DNA that bind to starting region of the DNA to be copied
Taq - Thermus aquaticus Polymerase is the polymerase enzyme isolated from a bacterium that can withstand high heat
Nucleotides – individual A, T, C, & G nucleotides in solution
Buffers – provide the proper chemical environment for annealing and polymerase to occur

19. PCR Product The product is the target region that is amplified.
In the first few rounds of replication the taq continues to copy beyond the end of the target
However, by the 3rd round of replication product containing only the target sequence is produced

21. Nested PCR Includes two rounds of PCR
First Round copies a larger region
And some extraneous regions
Second round copies the target
Used when ideal primers sequences are unavailable

22. rtPCR Reverse Transcriptase PCR uses RNA as a template, instead of DNA
Product contains transcribed genes, instead of genomic DNA
Lets you identify what genes are currently being used to make proteins

24. Today’s Lab Extract genomic DNA Set up first round of nested PCR
Amplifying a larger region
Lab II
Second, nested PCR Reaction
Lab III
DNA electrophoresis to look for results (product)

26. Lab II Today’s Lab - PCR Round II
Exonuclease – cleaves nucleotides from 5’ end of DNA
Used to remove erroneus/excess DNA from 1st PCR Reaction
Taq lacks proof-reading activity, can add erroneus nucleotides
Only treat samples with plant genomic DNA
Arabidopsis & Spinach

27. Nested PCR

28. Today’s Lab Treat samples with Endonuclease
Set up new PCR reactions with second set of primers
Load your samples into the thermocycler