Download PCR labwork 2 ENG

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
Sterile dH2O
10x Hot Fire DNA
buffer
MgCl2 (25mM)
dNTP mix (10mM)
Primer Fw (100 pmol/µl)
Primer
Rev
(100
pmol/µl)
Template DNA
Hot
FirePol
DNA
Polymerase (5U/µl)
Total
volume
of
reaction
PCR 1
volume,
µl
PCR 2
volume, µl
2.5
2.5
PCR 3,
NTC
volume, µl
17.4
2.5
Final
concentration
2.5
0.5
0.8
0.8
2.5
0.5
0.8
0.8
2.5
0.5
0.8
0.8
2.5mM
200 µM
0.5
0.5
0.5
50 ng
2.5U
25 µl
25 µl
25 µl
1x
 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 (A260), 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 A260/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
90 Fw
91 Rev
S19l Fw
S19l Rev
Sequence, 5’- 3’
AGG GAT GGA GCT TAC GAA CG
GCC CAC ACC ATG CCT TAC TCA
GCC ATA GCT CCC TGT CCC TCT CC
TTC CCC ATC GTC GTC CGT CTC GTC
Tm
3. What is the size of an amplified HNF1A and GCK4 PCR fragments?
__________________________________________________________________