Detection of a Human VNTR
Sequence Using Polymerase
Chain Reaction
Determining the Genetic Variability
of our Biology 22 Class
Schedule
• Day 1
– Isolate DNA from cheek cells
– Amplify VNTR sequences by PCR
• Day 2
– Agarose gel electrophoresis of PCR products
– Data Analysis
What Is PCR?
• PCR is a method for amplifying DNA.
• It requires the following components:
– Primers---determine the sequence to be
amplified
– DNA Polymerase ---Taq polymerase, stable at
high temperatures required by the reaction
– Nucleotides
– Ions, such as magnesium chloride
– Target sequence (supplied by your cheek cells)
Conditions for Polymerase Chain Reaction
to Detect D1S80 VNTR sequence
94oC for
30 seconds
72oC for
30 seconds
65oC for
30 seconds
D1S80 VNTR amplified by PCR
• VNTR= variable number of tandem repeats
• D1S80
– Found on Chromosome 1
– Contains 16 nucleotide sequence repeated 16-40
times
– Most individuals are heterozygous, having
different numbers of repeats in each of their two
D1S80 loci
Isolation of Cheek Cell DNA
• Do ALL steps listed on the laboratory handout (p.14-15)
– Collect cheek cells with cotton applicator. Transfer
cells to PBS solution. Transfer solution to screw cap
micro test tube. (1-4)
– Spin micro test tube to collect PELLET, remove
supernatant with pipette. (Steps 5-6)
– Add 100 ul well-suspended chelating agent to
PELLET and resuspend pellet. (steps 7-8)
Isolation of Cheek Cell DNA
• Do ALL steps listed on the laboratory handout (p.14-15)
– Place micro test tube in boiling water bath for 10
minutes. (Step 9). Cool and mix (Step 10)
– Spin micro test tube, remove SUPERNATANT
(containing cheek cell DNA) with pipette. Transfer
to clean tube labeled with your “class number”
(Steps 11-12)
– Keep DNA sample on ice. (Step 13)
Setting up PCR Reactions
• Follow Directions on laboratory handout (p.16):
– To a (very tiny!) tube with PCR bead, add 20 ul
D1S80 primer/solution (step 2)
(Instructor will complete this step)
– Add 5.0 ul of your cheek cell DNA to this tube
(step 2), label with your “class number”
– Mix and store this tube on ice prior to loading into
the thermal cycler (step 6)
Polymerase Chain Reaction
• Place your PCR tube in the Thermal Cycler
• It is programmed for 32 cycles of 94oC,
65oC, 72oC for 30 seconds each.
• After PCR, samples will be stored in the
freezer until the next laboratory period.
Gel Electrophoresis
• Gel electrophoresis
– Add 5 ul of 10x gel Loading solution to your
sample. (Step 7, p. 16)
– Incubate standard (prepared by your instructor)
and PCR sample for 2 minutes at 50oC
– Load entire volume of standard and sample
– Each gel should have one lane of standard and
5 student samples (make drawing of order by
class number)
Gel Electrophoresis
• Gel electrophoresis
– Run gel at 125 volts for 90 minutes or until
tracking dye has traveled 4.5 cm
– Bring gel to UV box for illumination and
photography
– Record your genotype for the D1S80 locus on
spread sheet
DNA Standards for
VNTR Insertion Analysis
Sizes of Standard Fragments in 200 bp increments
– Largest fragments will not be well resolved
– 1200 base pairs
– 1000 base pairs
– 800 base pairs
– 600 base pairs
– 400 base pairs
– 200 base pairs