PCR (Polymerase Chain Reaction)

1. PCR (Polymerase Chain Reaction)

2. What Does a PCR do? • Copying machine for specific parts of DNA. • Chain rxn. in which DNA template exponentially amplified • PCR makes billions of copies of specified DNA segment in few hours • No need to screen a DNA library for desired gene. • Bypasses need to use bacteria for amplifying DNA • Only need small amounts of DNA in starting material. • Helps when dealing w/ scant clues at crime scenes. • DNA can be partially degraded as long as some molecules contain complete target sequence. • Allows scientists to deal w/ ancient DNA.

3. Ingredients for PCR • Double-stranded DNA containing region to be amplified. • Heat-resistant DNA polymerase • Enzyme assisting in DNA replication. • Catalyzes polymerization of dNTP’s (nucleotides) alongside DNA strand. • Polymerization is a process of reacting monomer molecules to form polymer chains. • DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group on DNA segment, and therefore needs a primer at which it can add the first nucleotide. • If non-heat resistant polymerase used - protein would denature during 1st heating step and would have to be replaced after each cycle. • Key to automating PCR: discovery of heat-stable polymerase • Isolated from bacteria in hot springs.

4. Ingredients Continued • Two 15-20 nucleotide DNA strands that serve as primers. • One primer complementary to beginning of 3’ of the target DNA sequence. • Second primer complementary to the 5’ end of the sequence on the other strand. • Known as oligonucleotides. • Buffer solution • Provides suitable environ. for optimum activity and stability of polymerase.

5. Step 1: Denaturation (Separation) • Mixture heated to 94-98°C for 20-30 seconds. • Causes melting (denaturation) of DNA by disrupting the hydrogen bonds between complementary bases of the DNA strands. • Left w/ single strands of DNA.

6. Step 2: Annealing (H-bonding) • Rxn. temp. lowered to 50-65°C for 20-40 seconds. • Allows annealing of 2 primers to single-stranded DNA template. • Primers H-bond to ends of selected DNA sequence.

7. Step 3: Completing the Strands • DNA polymerase binds to the primer/single-strand DNA template and begins DNA synthesis. • Extends the primers in 5’ to the 3’ direction. • Completes single-stranded DNA starting from primer location. • Uses DNA building blocks- nucleotides (ATCG)-also known as dNTPs.

8. Step 4 and On • Repeat! • At first there’s exponential amplification • At every cycle, amount of product doubles • Then rxn. slows. • Polymerase loses activity and reagents (dNTPs+primers) begin to run out. • Final stage is the plateau. • No more product accumulates due to exhaustion of reagents and enzyme. • By end of 3rd cycle ¼ of the molecules are identical to target segment and both strands appropriate length • By cycle 30 99% of molecules match the target DNA sequence.

9. What’s This Good For? • Has had major impact on bio research and technology • Used to amplify DNA from wide variety of sources. • Detects small amounts of elusive infective agents in bodily fluids. • Can diagnose human genetic disorders by using primers that target the genes associated with these disorders • Ex. sickle-cell, hemophilia, cystic fibrosis. • Individuals identified before onset of symptoms and before birth. • Can identify symptom-less carriers of potentially harmful recessive alleles. • DNA amplified from fragments of ancient DNA from frozen wooly-mammoth • DNA from fingerprints or from tiny amounts of blood tissue found at crime scenes. • DNA of viral genes from cells infected with viruses. • Permits identification of non-cultivatable or slow-growing microorganisms. • Mycobacteria, anaerobic bacteria, or viruses from tissue culture and animals.