Back to : Contents . BioLab Help . Sample Experiments
In sickle-cell anaemia there is a point mutation in the beta-globin gene on chromosome 11 which codes for one of the proteins that is part of the haemoglobin molecule. This mutation replaces a thymine with a guanine in the sixth codon of the gene (i.e. GAG -> GTG), producing a valine instead of a glutamine at this position in the beta-globin protein.
The change in the DNA sequence means that the restriction enzymes DdeI and MstII which normally cut at this position can no longer cut here, and allows us to develop a diagnostic test.
This diagnostic test involves:
The primers were designed to amplify this region of the beta-globin gene starting 55 bp before the restriction site and ending 55 bp after the site. The PCR reaction therefore produces a DNA fragment 107 bp long. Digesting this DNA can give 3 possible results:
Using DNA from normal individuals (homozygous), who produce haemoglobin-A from normal beta-globin, the restriction enzymes cut the PCR fragment in half and a 55 bp band is seen on the gel.
Using DNA from an individual who has two sickle-cell alleles (homozygous) and produces haemoglobin-S, the restriction enzymes cannot cut the PCR fragment so a 110 bp band is seen on the gel.
Using DNA from an individual who is a sickle-cell carrier (heterozygous) and produces both haemoglobin-A and haemoglobin-S, both 55 bp and 110 bp bands will be seen on the gel.
Load experiment 10 into BioLab. It contains 8 samples.
The samples contain DNA fragments produced by PCR, using the primers described above, from DNA extracted from different individuals
Run the samples on a gel
Because the bands are small you may need to use a high percent agarose gel e.g.1.4% agarose and you may have to reduce the gel run time to get all of the bands on the gel.
If you want to check the size of the band click on the well of an unused track and choose the 50 bp ladder from the sample combobox.
You should see that each sample gives one band (110 bp), as shown in diagram 1.
Digest the DNA with either DdeI or MstII
Adjust the gel agarose concentration and/or run time to get all bands on the gel, and select the 50 bp ladder size marker.
Now you should see that the haemoglobin-A samples have been cut in half (55 bp)
but the haemoglobin-S samples
have not. A picture of the gel we obtained is shown in diagram 2. (Sample 1 has
been loaded in track 2,
sample 2 has been loaded in track 3, and sample 3 has been loaded in track 4.
all three samples have been digested with DdeI).
Use this procedure to determine whether samples 4-8 are from:
Click here to check your results!