Detection of a Human VNTR Sequence Using Polymerase Chain Reaction

1
Detection of a Human VNTR Sequence Using
Polymerase Chain Reaction

• Determining the Genetic Variability of our
  Biology 22 Class

2
Schedule

• Day 1
• Isolate DNA from cheek cells
• Amplify VNTR sequences by PCR
• Day 2
• Agarose gel electrophoresis of PCR products
• Data Analysis

3
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)

4
Conditions for Polymerase Chain Reaction to
Detect D1S80 VNTR sequence
5
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

6
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)

7
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)

8
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)

9
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.
  

10
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)

11
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

12
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