Student name, surname_____________________________________________________

Laboratory work Nr.2

Polymerase Chain Reaction (PCR)

The aim is to amplify from extracted genomic DNA HNF1A and GCK4 gene
fragments by PCR and evaluate PCR fragments size by agarose gel electrophoresis
method.

Introduction

The polymerase chain reaction is one of the most powerful in vitro laboratory
methods ever discovered. This method is very sensitive and specific with a great
degree of flexibility. PCR allows a single, short region of a DNA molecule to be
amplified to extremely high copy numbers using a simple set of reagents and a basic
heating and cooling (denaturing and annealing) cycle.

PCR requires the DNA template, DNA polymerase, free nucleotides dNTPs (dATP+
dCTP+ dGTP+ dTTP) mix. It also requires two unique single stranded DNA
oligonucleotide primers, which anneal to the regions upstream (5’) and downstream
(3’) of the DNA segment to be amplified. When these reagents are combined in an
appropriate buffer, a series of heating (denaturing) and cooling (annealing) steps
allow the DNA polymerase to copy the DNA in between the oligo primers. PCR
creates several micrograms of target DNA from just a few nanograms of template
DNA through several cycles of denaturation, annealing, and synthesis. After the PCR
is complete, the product can be verified based on size by gel electrophoresis .

Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g.
length in base pairs) for visualization and purification. Electrophoresis uses an
electrical field to move the negatively charged DNA toward a positive electrode
through an agarose gel matrix. The gel matrix allows shorter DNA fragments to
migrate more quickly than larger ones. Thus, you can accurately determine the length
of a DNA segment by running it on an agarose gel alongside a DNA ladder (a
collection of DNA fragments of known lengths).

Target substrates

Diabetes caused by mutations in the HNF1A (encoding hepatocyte nuclear factor-1

alpha) and GCK4 (encoding glucokinase 4) genes is one of the most common types of
maturity onset diabetes of the young (MODY). HNF1α is a transcription factor that is
important for the normal development of beta cells. Mutations in the HNF1A gene
cause diabetes by lowering the amount of insulin that is produced by the pancreas.
Mutations in HNF1A accounts for 70% of MODY cases.
GCK4 is a gene that codes for an enzyme, known as glucokinase, which helps the
body produce insulin in response to increases in blood sugar. Mutations in one of the
two copies of the GCK4 gene result in blood sugars that are mildly elevated above
normal levels. This is one of the most common forms of MODY, it is estimated to
affect about 1 in every 1000 people.

PCR procedure

I. Setting up the reaction mix

You will have 3 PCR reaction mixtures:

a. GCK4 gene fragment amplification

b. HNF1A gene fragment amplification
c. Negative control- no template control (NTC)

NTC control contains all reagents except DNA template. NTC is essential for
detecting contamination or non-specific amplification in your reaction.

1. Place 0.2ml PCR tubes on ice.

2. Set up 25 µl PCR reaction (keep all your reagents on ice).

Reagents PCR 1 PCR 2 PCR 3, Final

volume, volume, µl NTC concentration
µl volume, µl
Sterile dH2O 17.4
10x Hot Fire DNA 2.5 2.5 2.5 1x
buffer
MgCl2 (25mM) 2.5 2.5 2.5 2.5mM
dNTP mix (10mM) 0.5 0.5 0.5 200 µM
Primer Fw (100 0.8 0.8 0.8
pmol/µl)
Primer Rev (100 0.8 0.8 0.8
pmol/µl)
Template DNA - 50 ng
Hot FirePol DNA 0.5 0.5 0.5 2.5U
Polymerase (5U/µl)
Total volume of 25 µl 25 µl 25 µl
reaction

 In PCR1 tube the primers 90 and 91 should be used for amplification of the
GCK4 gene fragment.
 In PCR2 tube the primers S19L Fw and S19L Rev should be used for
amplification of the HNF1A gene fragment.
 In PCR3 tube you can choose one primer set or another.
 II. PCR reaction
3. Set up the PCR program in PCR thermocycler

PCR program:

a. Initial Denaturation for 10 minutes at 95°C: In this initiation step the

hydrogen bonds are broken between the nucleotide base pairs and DNA
strands separate from each other.
b. Denature 30 seconds at 95°C: Continued denaturation of DNA double helix.
c. Anneal primers for 30 seconds at 60°C: The forward and reverse primers
anneal to each of the single stranded DNA template strands. The DNA
polymerase bind to the primer DNA sequence.
d. Extend DNA for 30 seconds at 72°C: The Taq polymerase has an optimal
temperature around 70-75°C so this step enables the DNA polymerase to
synthesize and elongate the new target DNA strand accurately and rapidly.
e. Repeat steps 2-4 35 times.
f. Final Extension for 7 minutes at 72°C: A final extension to fill-in any
protruding ends of the newly synthesized strands.
4. Place the PCR tubes into PCR thermocycler and run the program.

A complete PCR reaction will be performed in a 2 hours.

III. Validating the PCR reaction

Once your PCR reaction has run, you will determine success or failure.

You will take some of the final PCR reaction and run it out on an agarose gel with an
appropriate molecular weight marker to make sure that the reaction was successful
and if the amplified product is the expected size relative to the maker.

5. Add 5 µl of 6x Loading dye into each PCR reaction tube and vortex.
6. Carefully load your samples into the wells of the 1% agarose gel and run the
gel.
Questions:

DNA yield and purity could be estimated by measurement of absorbance.

DNA concentration is estimated by measuring the absorbance at 260nm (A 260), adjusting the A260
measurement for turbidity (A320 measurement), multiplying by the dilution factor, and using the
relationship that A260 of 1.0= 50 µg/ml pure double stranded DNA.
The most common DNA purity calculation is the ratio of the absorbance at 260nm divided by the
readings at 280nm. Good quality DNA will have an A 260/A280 ratio 1.7-2.0. A reading below 1.7 does
not render that DNA unsuitable for any application, but lower ratios indicate more contaminants are
present.

1. What is the concentration and purity of your isolated genomic DNA?

Concentration ________________________________________

Purity_________________________________________________

If your DNA concentration is very low or DNA is absent, explain what possibly
could go wrong during DNA extraction procedure.

2. Calculate the melting temperature (Tm) of each primer by using formula

Tm = 2 X (A+T) + 4 X (G+C) (it will give you approximate Tm of your primers)

Primer Sequence, 5’- 3’ Tm

90 Fw AGG GAT GGA GCT TAC GAA CG
91 Rev GCC CAC ACC ATG CCT TAC TCA
S19l Fw GCC ATA GCT CCC TGT CCC TCT CC
S19l Rev TTC CCC ATC GTC GTC CGT CTC GTC

3. What is the size of an amplified HNF1A and GCK4 PCR fragments?

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