Amelogenin

Amelogenin is a gene that encodes a protein that is important for the formation of mammalian tooth enamel. Determining an individual’s sex based on DNA sequences is a highly reliable forensic analysis used to identify persons that are difficult to recognize. These people may be victims of incidents such as car crashes, fires, terrorists, or rape investigations (Nazari & Joshi, 2008). The Y chromosome comprises a molecular marker that is primarily used to determine the sex of unknown DNA samples. The amelogenin gene, which is important for distinguishing male and female from a DNA sample mixture, is found in all vertebrates. The amelogenin (AMEL) locus normally encodes for protein for the formation of the tooth enamel, and mutations in this gene normally lead to enamel condition known as amelogenesis imperfect which leads to the formation of an abnormal tooth enamel. The location of the AMEL locus consists of two homologous, ie, the AMELX located at the short arm of the X chromosome and the AMELY which is located near the centromere of the Y chromosome. The amelogenin protein is expressed on the sex chromosomes X and Y , but the level of their expression differ(Nazari & Joshi, 2008).To establish whether the PCR products were obtained from the male or female donors, the AMELX and AMELY are distinguished using short primers that targets the AMELX sequence that is are not always present in the AMELY. After the amplification of the AMELY locus and a shorter fragment of the AMELX are observed, it means that the sample is obtained from an individual having only the X chromosome and hence will display the female characteristics. However if two fragments of both lengths are observed, then it is a sample from an individual possessing both the X and Y chromosome and hence will display the male phenotype(Nazari & Joshi, 2008).In this practical, the DNA samples were obtained from cells in the mucosal lining. After extraction, the DNA samples were amplified through PCR, and the band images obtained showed that one has two bands while the other has only one band. The PCR product analysis indicated that the amelogenin gene has different versions in male and female regarding the number of nucleotide sequences and these differences are important in sex determination (Nazari & Joshi, 2008).This was expected from the practical and from the images it can be seen that one chromosome has only one fragment visible while the other one has two and hence the male and the female can easily be identified. The one with one fragment is the female while the one with two bands is from a male. 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. At times the AMELY portion may fail to amplify and hence making it impossible to determine sex of the DNA donor.

Question 2

a) The human chemokine receptor CCR5 normally act as a coreceptor with the CD4 as the entry sites for the HIV virus into the cells of the immune system. These receptors rest on the cell surface of the body cells and therefore allows for the binding of the HIV virus into the cells. A mutation on the CCR5 gene results in the formation of a short or a truncated protein that does not reach the cell surface and hence rendering it impossible for the binding of the HIV virus. The DNA to be obtained from the blood cells using the .The coding sequence of the CCR5 gene will then be taken 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 will then be 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 used should be sequence-specific which makes it able to hybridize with the targeted sequence. The DNA samples after amplification could also be taken through sequencing. In this case, the sequence alignment of the bases along the DNA will be analyzed and then compared to that of the wild-type. It will probably 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 materials were used to carry out the essay, 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) Analysis of DNA obtained from 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, the band images produced are not similar(Kordelas, Verheyen & Esser, 2014).The one obtained from an HIV positive individual indicates that the CCR5 sequence is present and hence giving up to 189 base pairs(Al-Jaberi et al., 2013).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 is a group of genes 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 due to a deletion removes a section of genes responsible for the formation of the protein that is 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.One of the alleles of such an individual will, therefore, have more bands as compared to the other This is due 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 be formed.

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).Individuals who take any mutation that results in the formation of an abnormal protein that is always required for the survival of any disease-causing pathogen will be able to resist that pathogen. The CCR5 is just an excellent example of such a gene (Al-Jaberi et al., 2013).It is required for the formation of proteins that act as the binding sites of the HIV and hence its absence due to a mutation leads to no structure of the binding site of the infection and consequently the individual will be able to resist the HIV (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, the presence of 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 who do not have such a mutation at all.

Question 4

The CCR5 gene is found on an autonomic chromosomes ie, the chromosome that is not a sex chromosomet.The presence or the absence of the gene on the DNA cannot 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 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, GA implies the individual is partially resistant to the HIV while the presence of an AA suggests the person is remarkably immune to the infection since the binding sites for the virus have not been formed(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