Introduction

After reading through our various papers on the CCR5 coreceptor, we thought it might be interesting if, as a group, we would see if any members had the CCR5 32bp deletion. We ran the experiment in Dr. Grimes' laboratory.

We amplified our DNA-(PCR) using primers specific for a small region of the CCR5 gene which contains the site of the 32bp deletion. Amplified DNA was then cut with a restriction enzyme and a run or an agarose gel to separate the fragments of this region of the gene. The DNA was visualized by ethidium bromide staining and a photograph is shown below.

Results

Please see The Process for the procedure.

The following picture shows the results of our DNA tests.

General Information a wild type individual has 735 base-pair fragments a heterozygous individual has 703 base-pair fragments The fragments were viewed on a 2% Agarose gel in TBE <- Click image to enlarge

Results from Left to right

1.      100 base-pair DNA ladder
2.      Tony PCR uncut (wild-type)
3.      Tony PCR ECOR1 digestion
4.      Margie PCR uncut (heterozygous)
5.      Margie PCR ECOR1 digestion
6.      Oscar
7.      Oscar ECOR1 digestion
8.      Thu
9.      Thu ECOR1 digestion
10.      SAC
11.      SAC ECOR1 digestion
12.      IUT
13.      IUT ECOR1 digestion
14.      SL
15.      SL ECOR1 digestion
16.      JF
17.      JF ECOR1 digestion
18.      SH
19.      SH ECOR1 digestion
20.      100 base-pair ladder

The gel shows that all students in the group are wild type, therefore homozygous for the 735bp fragments. However, our mentor was heterozygous for this gene as is seen in lanes #04 and #05 of this gel. In lane #04, the amplified DNA shows two bands, one at 735bp and one at 703bp. In lane #5, an ECO RI digestion of this DNA shows there are gene fragments as opposed to two in all the other digests. A wild type 735bp fragment is cleaved into two bands of 403 and 332 bp by ECO RI digestion. The mutant CCR5 703bp fragment is cleaved into two bands of 371bp and 332bp by ECO RI digestion. Therefore, as shown in lane #5, there are three bands representing digested DNA of 403,371, and 332 bp.

Method reference:

Samson, Michael et al. Nature. Vol. 382. pages 722-725. August 1996.