PCR Optimization Flashcards

Week 6 - PCR Optimization

Learning Objectives

1. Describe the purpose of each component in the polymerase chain reaction.

2. Predict how varying each component will impact PCR.

3. Relate DNA copy number to the mass of a DNA molecule.

4. Calculate the number of products generated during a PCR.

5. Understand the energetics and structural/functional anatomy of a DNA polymerase.

6. Understand the basic thermodynamics of the “hot start” mechanism.

7. Set up serial dilutions and master mixes.

PCR Components and Their Purposes

• Tris pH 8.4: Maintains optimal pH for enzyme activity.

  • Concentration: 200 mM

• KCl: Maintains optimal salt requirements for enzyme activity.

  • Concentration: 500 mM

• dNTPs (dATP, dTTP, dCTP, dGTP): Building blocks for new DNA molecules.

  • Concentration: 10 mM each

• MgCl2: Cofactor for DNA polymerase; stabilizes primer/template binding.

• Template DNA: Contains the DNA sequence to be amplified.

• Forward Primer: DNA polymerase extends this in the 5' to 3' direction on one strand.

• Reverse Primer: DNA polymerase extends this in the 5' to 3' direction on the other strand.

• DNA Polymerase: The enzyme that synthesizes new DNA strands.

Impact of Increasing PCR Component Amounts

10X Buffer, MgCl2, and dNTPs

• High potassium (from 10X buffer) stabilizes primer/template interactions.

• High magnesium stabilizes primer/template interactions.

• High dNTPs bind to magnesium ions, reducing free magnesium and destabilizing primer/template interactions.

Primer to Template Ratio Calculation

• Given Concentrations:

  • Forward Primer (SMOX): 0.5 \mu M

  • Plasmid DNA Template: 1 \times 10^7 copies

• Calculations:

  • Convert primer concentration to molecules:
    0.5 \mu M = 0.5 \frac{\mu Moles}{Liter} = 0.5 \frac{\mu Moles}{10^6 \mu Liters}
    0.5 \frac{\mu Moles}{10^6 \mu Liters} \times \frac{1 Mole}{10^6 \mu Moles} \times \frac{6 \times 10^{23} molecules}{Mole} = 3 \times 10^{11} molecules/\mu L

  • For a 20 μL reaction:
    3 \times 10^{11} \frac{molecules}{\mu L} \times 20 \mu L = 6 \times 10^{12} primer molecules

  • Ratio of primer molecules to template molecules:
    \frac{6 \times 10^{12} primer molecules}{1 \times 10^7 template molecules} = 6 \times 10^5

• There are 6 \times 10^5 primer molecules for every template molecule.

PCR Product Generation

• Each cycle doubles the number of DNA products.

• After n cycles, the number of products is 2^n.

• For a 40-cycle PCR:
  2^{40} = 1,099,511,627,776 \approx 1 \times 10^{12} products

• During a 40-cycle PCR, primers are eventually used up.

Annealing Step Binding Reaction

• Components: Forward Primer (FP), Reverse Primer (RP), Template (Temp.)

• Reversible binding reactions:
  FP + RP + Temp \rightleftharpoons FP/RP/Temp
  FP + RP \rightleftharpoons FP/RP
  FP + Temp \rightleftharpoons FP/Temp
  RP + Temp \rightleftharpoons RP/Temp

Impact of Increasing Primer Concentration and Template DNA Copy Number

• Increasing primer concentration increases primer/primer interactions, leading to primer dimers.

• Increasing template DNA copy number increases PCR products until primers or polymerase are depleted.

• Increasing either/both can sequester Mg^{2+} ions, destabilizing primer/primer and primer/template interactions.

Mass Calculation of Plasmid DNA

• Given: 10^7 plasmid DNA molecules, 5 kb size

• Approximation: 10 pg of 10 kbp \sim 10^6 copies, therefore 5 pg of 5 kbp \sim 10^6 copies

• Therefore, 50 pg of 5 kbp \sim 10^7 copies

Taq DNA Polymerase Reaction

• Catalyzed Reaction: DNA polymer + dNTP → DNA polymer(+1) + PPi

• The reaction is

In this lab, you will optimize a PCR that amplifies a region of the SMOX cDNA insert that is in the pBluescript plasmid you isolated in a previous lab. You will determine the conditions yielding the best PCR product. The primers you'll use today are similar to those you designed last period. Note that the SMOX CDNA is cloned in the multiple cloning site (MCS, or polylinker) of pBluescript SK (+). You will be setting up PCR reactions. Unlike the first week of lab, this time you will be adding all the components yourself.

10X Buffer

10 mM dNTPs

25 mM MgCl2

10 µM Forward primer

10 µM Reverse primer

Taq polymerase

Template

Water

ampicillin

pBluescript SK+ 3.0 kb

fl (+) ori

clacz

Kpn I

MCS

Sac I

Plac

PUC ori

Imagine if you had to digest 90 different templates with the same set of restriction enzymes, and so you went about setting up 90 individual restriction digestions. That could involve up to 630 different pipetting steps and lots of chance for human error. Master mixes can be used to save time and dramatically reduce the amount of pipetting and resultant errors and inconsistencies. The general idea is to pool all of the elements that reactions have in common. For example, in the 90 restriction digests described above, these common elements are water, buffer, BSA, and enzymes. To make a master mix, you would pool enough reagents for 90 reactions into one large tube and then divide this bulked reaction mix into 90 individual aliquots in separate tubes, to which you could then add the individual templates for testing.

To set up a table for calculating how to make a master mix, you first set up the components that are necessary for one reaction. Then you multiply each of the components (except the one component that does not go into the master mix, in this case, DNA) by the number of reactions plus a fudge factor.

For example, to digest 10 µg of DNA with EcoR1 in a volume of 100 \mu L, set up a standard restriction digest table.

Multiply the common components that will go into the master mix by 90, plus a fudge factor (10 extra reactions, in this case) for a total multiplication factor of 100. This will allow some pipetting error such that you are not shorted in the last few reactions.

To a tube large enough to hold the entire volume, add the water, 10X buffer, BSA, and EcoR1 to form the master mix. Then, before you divide up your master mix, you first will want to aliquot 10 \mu L of your 90 different DNA samples into 90 different tubes using a fresh, sterile pipet tip for each DNA sample/tube. Then aliquot 90 \mu L of master mix into each of your different tubes with DNA in it, again using a fresh, sterile pipet tip for each 90 \mu L aliquot. This results in 185 pipetting steps instead 630 pipetting steps. If each pipetting step takes 10 seconds, you will save about 75 minutes of pipetting. Please note that you can set up master mixes for a variety of reactions including PCR (e.g., today's experiment).

Calculating template number:

The SMOX cDNA insert is approximately 3.2 kb making the entire template for PCR (plasmid with insert) approximately 6kb in length. When amplifying from a plasmid, you want to add approximately 10^7 copies to use as template for PCR.

A general rule is: 10 pg of a 10 kb molecule is approximately 10^6 copies of the template. (And 6 pg of a 6 kb molecule is ~ 10^6 copies, and so on…)

Now you know your plasmid concentration. Next you will have to calculate the volume of plasmid that will yield 10^7 copies of template.

Will you be able to accurately pipette this volume? If not, you should dilute the plasmid so that it has a low enough concentration to allow accurate pipetting. For this exercise, the lowest volume you pipette should be 1 \mu L. (Keep your dilutions in microfuge tubes….this means that you may have to perform multiple dilutions - one of which will be 10^7)

B. (Work in pairs) Create a thermal cycling profile that takes into consideration the primer T and the product size.

PCR primer information:

Forward primer: SMOXF1 T 62.8^\circ C

5-GCA GGT GGT CAA GCG TTT GTT AGC AC-3'

Reverse primer: SMOXR1 T 62.6^\circ C

5-CCA GCG GAT