Amelogenin

Amelogenin and Sex Determination

Amelogenin is a gene that encodes a protein that is important for the formation of mammalian tooth enamel (Nazari & Joshi, 2008). The AMEL’s geographical positioning This protein’s gene is found on both the X and Y chromosomes and is found in all vertebrates. DNA samples were collected from cells in the mucosal lining in this reality. Following extraction, the DNA samples were amplified using PCR, and the band images revealed that one has two bands and the other has just one band. The PCR product analysis indicated that the amelogenin gene has different versions in males and females regarding the number of nucleotide sequences and these differences are important in sex determination. This is was expected from the practical and from the images it can be seen that one chromosome has only one band while the other one has two and hence the male and the female can easily be identified. This method is however not 100% accurate due to the high rates of mutations in the population. In some cases, for example, the amelogenin on the Y chromosome may have been deleted due to such mutations and hence causing a misidentification of such individuals as females.

Question 2

a) The human chemokine receptor CCR5 normally act as a co-receptor with the CD4 as the entry sites for the HIV virus into the cells of the immune system. The DNA to be obtained from the blood cells and then amplified through polymerase chain reaction to obtain a substantial amount of the sequence for proper and accurate analysis (Al-Jaberi et al., 2013).The amplified sequence is then run on a blotting paper forming dot- blots which then hybridize with a short inflorescent probe making it possible to determine the presence or absence of a CCR5 mutation in an individual. The probe should be sequence-specific which makes it to hybridize with the targeted sequence. The DNA samples amplified then sequenced. The sequence alignment is then analyzed and compared the wild-type. It will indicate that the alleles with 189 bp show a wild-type allele while that with 157 bp is a mutated CCR5 where 32 bp have been deleted. The following were used, 1.5ml microtube for collecting the buccal cell, 0.2-ml PCR microtube, 2–200 µL micropipette tip, 2–20 µl micropipette(s), PCR premix, microtubes (48 µl per tube), 3% w/v agarose gel, Tris about 89mm. hydrochloric acid, boric acid.

b) The DNA samples of two individuals where one has been exposed to the HIV but remains negative and a DNA sample of an HIV positive individual when taken through gel electrophoresis. An individual who can remain negative even after several exposures to the virus implies that the individual is resistant to the virus and does not have the binding sites for the HIV virus since it must enter the host cells and use the host cell machinery for replication. Since the CCR5 gene is absent in such individuals, the band images produced from electrophoresis are not similar. The one obtained from an HIV positive individual indicates that the CCR5 sequence is present and hence giving up to 189 base pairs. The sample obtained from a negative individual who has however been exposed to the virus but has shown a resistance indicates that the base pairs are only 157 meaning there are bases missing. A specific protein performing a specific role is therefore not being produced.This explains why someone may be HIV positive while the other does not have despite the various exposures to the virus. The mutation removed 32 bases representing a gene for the formation of the protein, the binding site for the HIV.

c) An individual carrying one copy of the mutant gene means that the individual is in the heterozygous for the condition.The person is therefore carrying the trait but however appears normal interms of the formation of the CCR5 receptor proteins.A good number of the receptors will be formed normally and some will be abnormally formed, either too short to carry out their function as receptors for the HIV virus and may not even be reaching the cell surface ie may be truncated proteins.When the DNA sample of an individual is analysed through gel electrophoresis,the allele fragments cannot be the same in length. One of the alleles will have more bands and will be much longer than the other one. This is because in heterozygous individuals, one of the alleles is mutated through deletion that leads to the loss of up to 32 base pairs in them, and hence the expression of the receptor for the protein where the HIV binds cannot take place.

Question 3

Despite the several exposures to HIV, some individuals remain seronegative to the HIV-1 infection. Such people carry a mutation in the CCR5 gene (Kordelas, Verheyen & Esser, 2014). Having one copy of such a mutation protects cells partially against HIV infection. For the virus to gain entry into the cells, it must bind to the receptor site is known as the CCR5 that usually is a co-receptor to the CD4 cells. It is mainly found on the surface of the immune cells. The mutation in the gene that encodes the CCR5 protein results in the formation of a defective receptor site that blocks the entry of the HIV into the body cells. When someone has copies of the defective gene, the individual will be completely resistant to the HIV and hence cant is affected even after continued exposure to the virus. Such individuals are said to be homozygotes for that trait. People who are heterozygous for the trait also show some resistance than those who do not have it at all. The heterozygotes express mostly abnormal CCR5 receptors on the surface of the cells and therefore affecting the development of the virus and hence a reduced progression of the HIV into full-blown for about two to three years (Al-Jaberi et al., 2013).Heterozygous individuals, however, have almost half of their CCR5 receptor being healthy and a half being poorly formed or truncated and therefore cannot be found on the cell surface. Such individuals thus present few receptors for the HIV binding on their immune cells such as macrophages (Kordelas, Verheyen & Esser, 2014).Given the fact that the HIV replicates very fast, few receptors means that its survival is much reduced and hence people with the heterozygous conditions will always show a reduced susceptibility to the HIV compared to those lack it at.

Question 4

The CCR5 gene is found on an autosomic chromosomes ie, the chromosome that is not a sex chromosome and is found on the body cells.The presence or the absence of this gene on the DNA cannot therefore be used to determine the sex of an individual.This is because sex determination generally involve the X and Y chromosome(Kordelas, Verheyen & Esser, 2014).Unlike the amelogenin gene that is located on both the sex chromosome X and Y and hence can be used to determine the sex of an individual by just looking at the number of electrophoretic products.

Question 5

To determine point mutations in a DNA sample, sequencing can be used as a way of identifying an unknown base at a particular section. The sequence of the unknown sample is run by the Sanger method or any other convenient method (Nazari & Joshi, 2008).The sequence of the unknown sample is then aligned and compared to the sequence without the point mutation, i.e., the wild-type.When the sequence of the unknown sample is compared against the sequence of the wild form, the point mutations can be easily identified, and the nucleotide bases which are affected by the modification can be easily identified.

The presence of GG genotype which means the individual is very susceptible to HIV.Such an individual has almost all the receptors for the HIV virus to bind and hence the virus has higher chances of survival as it easily enter into cells.After entering the cells ,it takes over the cells replicating machinary to replicate itself and increases in number.The higher their number, the more the host cells are destroyed and the immune system become compromised. GA implies the individual is partially resistant to the HIV because there are almost equal numbers of the normal receptors for the HIV virus and the abnormal ones.The presence of one mutant allele in such individuals therefore gives them an advantage over those that do not have the mutation.This is because there cell surface receptors specific for the binding of the HIV virus are few and this lowes rhe progression of the virus.The presence of an AA in an individual however suggest that person is remarkably immune to the infection.This is because the receptors specific to the binding of the virus are truncated and cannot reach the cell surface.The HIV cannot therefore enter the cells of such individuals making them highly resistant to the virus.(Nazari & Joshi, 2008).

There is a vast difference between an individual with the genotype GA and the one with the genotype AA in HIV resistance. An individual in the heterozygous condition is has got partial resistance to HIV infection while the one having the homozygous state will have strength over HIV infection since the recognition sites for the HIV are not formed and hence reducing the entry of the virus into the host cells.

Question 6

The use of target-specific probe can be used to identify the presence or the absence of a DNA sequence. The CCR5 D32 mutation is a result of a 32 base pair deletion on the DNA segment. When there is only one copy, the radioactive probe will just be visible at one point in the genome(Nazari & Joshi, 2008).The other with no copies of the mutation will show the probes at two points indicating the gene is present.

When an individual has two copies of the CCR5 mutation, the probe may not be seen because the sequences will not be detected, but the one carrying zero copies of the mutation will show two probes.

Two probes will be visible at a specific location on the DNA indicating the absence of the bases the particular area of the genome.

Question 7

Fredrick Sanger developed the Sanger sequencing technique in 1977.

Advantages

1. It helps in targeting small genomic regions in a large number of samples and hence its accuracy or efficiency is very high compared to other methods.

2. It helps in the sequencing of the variable regions of the genome and therefore providing a more in-depth coverage of the genome.

3. It assist in validating the results obtained from other methods like the next generation sequencing due to its efficient nature

4. Help in verifying plasmid sequences, inserts and mutations and hence making it very suitable for understanding the genome segment during sequencing.

5. It also helps in accurate genotyping of genetic microsatellite markers and therefore making it easier to understand the genome and make interpretations.

6. It helps in identifying single disease-causing genetic variants and thus making it a useful method for genome sequencing.

7. It can be used to sequence long or large-sized genomes at high accuracy levels as compared to the other ways

8. In the Sanger technique, fewer dangerous chemicals are usually used and hence it is a safer method of sequencing.

9. The gels or the autostrada commonly used remain clear and cleaner and thus reading the base is much more comfortable as compared to the other methods.

10. It is also more amenable to automation as compared to the maxam Gilbert method.

Disadvantages

1. It can sometimes be affected by nonspecific primer binding which can interfere with an accuracy of the procedure.

2. It can be affected by the formation of DNA secondary structures which hinder with the fidelity of the process.

3. It is very expensive to carry out as compared to the other methods of sequencing like the next generation sequencing.

4. Detection and separation of the samples remain labor intensive making the technique more cumbersome than the different methods.This makes it inappropriate in dealing with large examples involving up to a thousand patients

5. Labeling using the radioactive isotopes remains difficult dangerous and expensive.

6. At times the method is clumsy, time-consuming and unreliable and hence may be difficult in dealing with large samples

7.The data produced sometimes can be a low quality which is not worth the effort involved and therefore may not at times give accurate, expected results and accordingly when dealing with large samples, it may be difficult to obtain good results.

References

Nazari, R., & Joshi, S. (2008). CCR5 as Target for HIV-1 Gene Therapy. Current Gene Therapy, 8(4), 264-272. http://dx.doi.org/10.2174/156652308785160674

Kordelas, L., Verheyen, J., & Esser, S. (2014). The shift of HIV Tropism in Stem-Cell Transplantation with CCR5 Delta32 Mutation. New England Journal of Medicine, 371(9), 880-882. http://dx.doi.org/10.1056/nejmc1405805

Al-Jaber, S., Ben-Salem, S., Messedi, M., Ayadi, F., Al-Gazali, L., & Ali, B. (2013). Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis. Gene, 529(1), 113-118. http://dx.doi.org/10.1016/j.gene.2013.07.062

Nazari, R., & Joshi, S. (2008). CCR5 as Target for HIV-1 Gene Therapy. Current Gene Therapy, 8(4), 264-272. http://dx.doi.org/10.2174/156652308785160674