Detection of the human VNTR using PCR*

*A Polymerase Chain Reaction Experiment

The experiment is divided into 2 parts

Isolate human DNA from cheek cells The DNA will be amplified using PCR

The DNA will be analyzed using gel electrophoresis

Polymerase Chain Reaction: another method of DNA amplification

Basis of PCR

Create conditions “in vitro” for DNA replication.

Requirements for replication

DNA template Primers Taq DNA Polymerase** Nucleotides MgCl++

Temperature cycles plays a key role:

Unwinding DNA Annealing Extension

98C 45-60C 72C

http://www.dnalc.org/Shockwave/pcranwhole.html

Primers determine

The sequence of DNA that will be amplified.

Basis for sequence specific amplification

•Two primers are used to bracket the area you want to amplify.

•Primers are single stranded synthetic sequences of DNA normally 20-30 bp.

•One primer is complementary to the beginning of the target gene on one strand while the other primer is complementary to end of the target gene on the complementary strand.

Summary:

Unwinding DNA Annealing Extension

98C 45-60C 72C

http://www.dnalc.org/Shockwave/pcranwhole.html

The experiment is divided into 2 parts

Isolate human DNA from cheek cells The DNA will be amplified using PCR

The DNA will be analyzed using gel electrophoresis

Highlights of Key Steps of your DNA Isolation

Each person should obtain

Cotton swabs

A test tube with 2 mls of buffer

A screw top microfuge tube

2 transfer pipets

Isolation of DNA

Cheek Cells

Obtain cells by swabbing the inside of the mouth with a cotton tipped applicator.

Next…

Place cotton head in 2 ml of PBS (in conical tube)

Swirl vigorously for 1 min. to dislodge cells

Press the cotton head against the walls to squeeze out as much liquid as possible

Use new applicator and repeat the above steps.

Next…

Transfer 2 ml to screw top tube.

Spin at 5000 rpm for 1 min. Be sure you have pellet. If not repeat swabbing.

Remove supernantant but SAVE PELLET (these are your cells!)

Add 100 ul of chelating agent to sample

Next…

Re-suspend the pellet in 100 ul of chelator solution: MAKE SURE PELLET IS RESUSPENDED!

Place screw top microfuge tube in boiling waterbath for 10 minutes to break (lyse) cells.

Next:

After 10 min boiling allow tube to cool (2 minutes and then spin microfuge tube for 2 minutes (5000 rpm)

Next:

Obtain a PCR tube with “PCR bead”

Add primers to bead and 5 ul of your supernatant and gently mix.

Label your PCR tube your assigned seat number

Place in PCR!

Summary of PCR tube

To PCR tube containing “bead” add:

20.0 ul of primer solution 5.0 ul of cheek cell DNA

VNTR : PCR reaction 32 cycles at:

94C 65C 72C

30 seconds 30 seconds 30 seconds

The gel electrophoresis

Analysis of your results will be carried and provided to you (time may not be available at the next lab for gel electrophoresis).

After PCR

Gel electrophoresis Warm samples (including ladder) 2 minutes at 50

C Load 25 ul of the sample Load 25 ul of ladder