Tina Doss Applied Biosystems PCR Tina Doss Applied Biosystems Colleen Malone/Jennifer Lockwood NPHS

Review- DNA Function DNA has two primary purposes: To make copies of itself so cells can divide and carry on the same information. To carry instructions on how to make proteins. 2

Review- DNA Packaging Approximately 3 billion base pairs in a single copy of human genome Chromosome = dense packet of DNA wrapped around proteins called histones

Review- DNA in Cells Somatic (body) cells are diploid = 2 sets of each chromosome 23 pairs or 46 chromosomes total 22 pairs of autosomes and 1 pair of sex chromosomes

Review - DNA Nucleotide DNA has 3 parts: phosphate group Nitrogenous base sugar (pentose) Click picture for DNA Structure animation

Nitrogenous Bases Pyrimidine = Single Ring T and C + Uracil (not shown) Purines = Double Ring A and G Sugar Sugar Adenine (A) Thymine (T) Nitrogenous Bases Sugar Sugar Guanine (G) Cytosine (C)

YOUR dNTP’s -The building blocks of DNA synthesis!

Review – “Backbone” of DNA Nucleotides are joined together by phosphodiester linkages (between phosphate of one nucleotide and the sugar of the next) This results in a backbone with a repeating pattern of: sugar-phosphate-sugar-phosphate... Phosphate 8

Remember: Dehydration Synthesis forms this bond (also known as a Condensation Reaction) During this linkage: Two phosphates are removed from your nucleotide triphosphate Water is formed as well Phosphate 9

Review- “Steps” of the DNA Ladder Complementary base pairing is completed through hydrogen bonds between bases: A=T (2 hydrogen bonds) G=C (3 hydrogen bonds) Chargaff’s Rule The strands run anti-parallel _ Click picture for Replication Fork animation

Review - DNA Replication A DNA sequence is written and read from 5’ to 3’. DNA Polymerase III is the enzyme that will “write” sequences. Much like we read in a certain direction, like left to right. Click picture for DNA Replication animation

Review- DNA Replication DNA Polymerase III follows base pairing rules to attach the new nucleotides to the growing strand: A=T (2 hydrogen bonds) G=C (3 hydrogen bonds) Chargaff’s Rule! Remember: The strands run anti-parallel _ Click picture for Replication Fork animation

Basic Vocabulary DNA may be denatured (denaturation) (split 2 strands) by: Heat DNA to near boiling temperatures Place DNA in a salt solution of low ionic strength Expose DNA to chemical denaturants (ex: Urea) -Chemical denaturants like urea and formamide form hydrogren bonds with the nucleotides preventing association with complementary DNA Strand.

Basic Vocabulary Denaturation is reversible. Process of 2 complementary DNA strands coming back together is called renaturation or reannealing. In lab, renaturation occurs during cold cycles. -Chemical denaturants like urea and formamide form hydrogren bonds with the nucleotides preventing association with complementary DNA Strand.

The Polymerase Chain Reaction (PCR)  defined: a technique that involves copying short pieces of DNA and then making millions of copies in a short amount of time. Advantages: Target DNA fragments to amplify Lots of DNA in short amount of time Can help identify small differences among individuals or populations (lots of applications use this technology: forensics, barcoding, medical research,….)  

BILLIONS of copies of DNA are produced in just a few hours In 6 cycles of PCR: cycle 1: 2 copies cycle 2: 4 copies cycle 3: 8 copies cycle 4: 16 copies cycle 5: 32 copies cycle 6: 64 copies cycle 20: 1,048,576!!

What do you need: (the “master” mix) 1. DNA fragment to be copied 2. Nucleotide Triphosphates ( all four dNTP’s) 3. DNA Primers (forward and reverse) need the 2 primers to “flank” the region of DNA to be copied. Use a forward and reverse DNA primer to begin at the starting point to isolate the target DNA sequence. (small DNA segments)

What do you need: 4. Taq polymerase (DNA polymerase isolated from bacteria-Thermus aquaticus, living in hot springs…their enzymes can withstand high temps!) 5. Reaction buffer – stabilize the pH 6. MgCl2 is needed for activation of the polymerase (co-factor; mineral needed to help enzyme function)

Basic Steps of PCR: 1)  Heat to Denature (separate) DNA strands-break H-bonds (95ºC) (~ 15 seconds) 2) Annealing: Cool to allow primers to bind (55ºC) (~30 sec) 3) Extension: Heat slightly so that Taq polymerase extends from the 3’ end of each primer (72ºC) (~40 sec - 2 min) (NOTE: Times of each step can vary depending on size of DNA that is amplified) ***Repeat steps #1-3 many times!!! --Let’s watch a video! http://www.youtube.com/watch?v=2KoLnIwoZKU This three-temp cycle takes about 2- 5 minutes so 35 cycles will take ~3 hours to complete!

You will also need equipment: 1) The instrument that heats and cools a DNA sample for PCR is called a Thermal Cycler. -Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems. -The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second. 21

Thermal block heats and cools to repeat the steps in PCR (20-50 cycles). 2. PCR tubes. -Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems. -The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second. 22

– PCR ∞ Basic Thermal Cycling Parameters: 95oC 10:00 72oC 55-65oC 0:15 0:30 0:40 4oC ∞ 1 HOLD 32 CYCLES 2 HOLDS Activation Denature Extension Final Extension Annealing -Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems. -The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second. This stage is to allow sufficient time for all DNA fragments from previous cycles to finish extension. 23

– PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 55-65oC 0:15 0:30 0:40 4oC ∞ 1 HOLD 32 CYCLES 2 HOLDS Activation Denature Extension Final Extension Annealing Final Hold -Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems. -The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second. This stage is to slow down all the processes and help keep the solution stable. This is like putting your sample in the refrigerator. 24

Why PCR? 1. Genomic DNA (target a region- i.e. DNA barcoding= 650 bp region of mitochondrial DNA) 2. Known DNA Fragment (DNA purification or make lots of copies to run other tests)