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.

Approximately 3 billion base pairs in a single copy of human genome

Chromosome = dense packet of DNA wrapped around proteins called histones

Review- DNA Packaging

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 in Cells

DNA has 3 parts: phosphate group Nitrogenous base sugar (pentose)

Click picture for DNA Structure animation

Review - DNA Nucleotide

LE 16-8

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

Sugar

Sugar

Sugar

Sugar

Purines = Double RingA and G

Pyrimidine = Single Ring T and C + Uracil (not shown)

Nitrogenous Bases

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

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

Review – “Backbone” of DNA

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

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

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Review- “Steps” of the DNA Ladder

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

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Review- DNA Replication

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)

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.

Basic Vocabulary

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 pH6. 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.

• Thermal block heats and cools to repeat the steps in PCR (20-50 cycles).

• 2. PCR tubes.

– PCRBasic Thermal Cycling Parameters:

95oC

10:00

95oC72oC 72oC

55-65oC0:15

0:30

0:40 10:00

4oC

∞

1 HOLD 32 CYCLES 2 HOLDS

Activation Denature Annealing Extension

Final Extension

This stage is to allow sufficient time for all DNA fragments from previous cycles to finish extension.

– PCRThermal Cycling Parameters:

95oC

10:00

95oC72oC 72oC

55-65oC0:15

0:30

0:40 10:00

4oC

∞

1 HOLD 32 CYCLES 2 HOLDS

Activation Denature Annealing ExtensionFinal Extension

Final Hold

This stage is to slow down all the processes and help keep the solution stable. This is like putting your sample in the refrigerator.

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)