Presentation on theme: "Tina Doss Applied Biosystems"— Presentation transcript:
1 Tina Doss Applied Biosystems PCRTina DossApplied BiosystemsColleen Malone/Jennifer LockwoodNPHS
2 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
3 Review- DNA PackagingApproximately 3 billion base pairs in a single copy of human genomeChromosome = dense packet of DNA wrapped around proteins called histones
4 Review- DNA in CellsSomatic (body) cells are diploid = 2 sets of each chromosome23 pairs or 46 chromosomes total22 pairs of autosomes and 1 pair of sex chromosomes
5 Review - DNA Nucleotide DNA has 3 parts:phosphate groupNitrogenous basesugar (pentose)Click picture for DNA Structure animation
6 Nitrogenous Bases Pyrimidine = Single Ring T and C + Uracil (not shown)Purines = Double RingA and GSugarSugarAdenine (A)Thymine (T)Nitrogenous BasesSugarSugarGuanine (G)Cytosine (C)
7 YOUR dNTP’s-The building blocks of DNA synthesis!
8 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...Phosphate8
9 Remember: Dehydration Synthesis forms this bond (also known as a Condensation Reaction)During this linkage:Two phosphates are removed from your nucleotide triphosphateWater is formed as wellPhosphate9
10 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 RuleThe strands runanti-parallel_Click picture for Replication Fork animation
12 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
13 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
14 Basic VocabularyDNA may be denatured (denaturation) (split 2 strands) by:Heat DNA to near boiling temperaturesPlace DNA in a salt solution of low ionic strengthExpose DNA to chemical denaturants (ex: Urea)-Chemical denaturants like urea and formamide form hydrogren bonds with the nucleotides preventing association with complementary DNA Strand.
15 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.
16 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 amplifyLots of DNA in short amount of timeCan help identify small differences among individuals or populations (lots of applications use this technology: forensics, barcoding, medical research,….)
17 BILLIONS of copies of DNA are produced in just a few hours In 6 cycles of PCR:cycle 1: 2 copiescycle 2: 4 copiescycle 3: 8 copiescycle 4: 16 copiescycle 5: 32 copiescycle 6: 64 copiescycle 20: 1,048,576!!
18 What do you need: (the “master” mix) 1. DNA fragment to be copied2. 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)
19 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 pH6. MgCl2 is needed for activation of the polymerase (co-factor; mineral needed to help enzyme function)
20 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!This three-temp cycle takes about 2- 5 minutes so 35 cycles will take ~3 hours to complete!
21 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
22 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
23 – PCR ∞ Basic Thermal Cycling Parameters: 95oC 10:00 72oC 55-65oC 0:15 0:300:404oC∞1 HOLD32 CYCLES2 HOLDSActivationDenatureExtensionFinal ExtensionAnnealing-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
24 – PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 55-65oC 0:15 0:30 0:404oC∞1 HOLD32 CYCLES2 HOLDSActivationDenatureExtensionFinal ExtensionAnnealingFinal 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
25 Why PCR?1. Genomic DNA(target a region-i.e. DNA barcoding= 650 bp region of mitochondrial DNA)2. Known DNAFragment(DNA purification or make lots of copies to run other tests)