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

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

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

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

Complementary Base Pairing

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

Nested PCR

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