A polymerase chain reaction (PCR)

A polymerase chain reaction (PCR) is a molecular procedure used in molecular diagnosis to amplify duplicate or copies of DNA fragments in a specific sequence of DNA (Garibyan and Avashia 2013, p.6). The procedure has proven to be effective in medical diagnosis. This procedure was used in this medical study to determine why excessive hair pigmentation happens in certain people. Although red hair pigmentation and fair hair or red pigmentation are not normal in people, it has been shown in many cases that they are caused by a mutation of the wild-type gene responsible for melanin formation. The colors of the hair are due to the effects of the melanin pigmentation. While melanin is categorized into eumelanin and phaeomelanin, each component determines different color pigment (Meredith and Sarna 2006, p.574). The brown or black color of the hair is due to the effects of the eumelanin while the reddish brown color of the hair is a result of the phaeomelanin (Ito et al. 2011, p.605). Despite the fact that the eumelanin and the phaeomelanin are component of the melanin, it is worth noting that one of the the causes of the difference is mutation at the MC1R gene.

The change in the color of the hair is due to the mutation of the MC1R gene responsible for the production of the melanin. MC1R is the gene which produces the protein known as the MC1R receptor or melanocortin 1 (Lalueza et al. 2007, p.1455). The protein then converts the phaeomelanin to the eumelanin (Simon et al. 2009, p.564). Mutations of the MC1R gene hinders the conversion of the phaeomelanin to the eumelanin and due to the frequent formation of the former, it builds up in the pigment cells and thus the red hair color. According to the patients sampled for the diagnosis, patient 1showed homozygous wild type. Patient 2 experienced Irish type of mutation at the Arg and Cys positions and this resulted in red hair. On the other hand, patient 3 experienced common type of mutation at the Val and Leu to form fair hair.

Methods

The main method used in this practical is the polymerase chain reaction (PCR) method. PCR is an in vitro scientific method that is used in the amplification of the genes obtained from the body tissues. In this report, the tissue source was hair (Maheaswari, Kshirsagar and Lavanya 2016, p.128). The components of this method include the DNA template, nucleotides, DNA polymerase and the primers. The template DNA is the sequence of the target DNA that needs to be amplified. The MC1R gene was used as the DNA template for amplification.

For designing the forward and the reverse primer, the genome of the MC1R gene was first identified using the NCBI webpage. After this, the primer was designed with an annealing temperature of 57oC which lasts for 30 seconds. The primer pair then amplified the exon parts of the MC1R gene. Based on the reverse and forward primers, the following parameters were used in the PCR method. The DNA template from the MC1R was first subjected to initialization temperature at 94oC for 5 minutes. The DNA template being in double strand, it was then subjected to high temperatures of 94oC for the 30 seconds. The high temperatures was to separate the double strands to single strands for annealing of the primer to the MC1R gene at 570C for 30 seconds. The oligonucleotide primer cannot anneal to the DNA template at the initial high temperatures and must be allowed to cool to 57oC for the formation of the double strands again. On the other hand, increasing the PCR specificity of the PCR requires increased temperatures to 70oC for maximum yield of the hybrid DNA.

The primed sequence of the MC1R gene was then allowed to undergo extension at 70oC in a thermocycler, which is a favourable temperature for the Taq polymerase functioning. The binding of the Taq polymerase to add the dNTPs took place in the 5’ to 3’ direction. After, the extension, the hybridized DNA underwent final elongation for 700C for 5 minutes and this took place in 30 to 34 cycles. At the final hold, the temperature was lowered to 4oC for a considerably long time.

DNA Isolation

First and foremost, the extraction solution of 400ul was put in place and rinsed. The Buccal swab was then collected from the cheek and placed into the extract solution. The mixture of the swab and the extract solution was then vertexed and incubated at 65oC. The tube was then transferred to a water bath at 98oC and incubated and vertexed for 2 minutes. The content was then transferred to a thermo cycler for amplification.

PCR analysis and Gene clean

After the amplification, the DNA solution was then purified using a QIAquick PCR purification kit before gel electrophoresis. A Buffer solution was added to the PCR sample and allowed to mix thoroughly. The pH indicator was then added to the solution. The DNA was to be analyzed using the gel electrophoresis and hence 1 volume of a loading dye was added to 5 volumes of the DNA solution already purified. The solution was the mixed before loading to the gel electrophoresis.

DNA sequence analysis

The DNA sequence was then analyzed for loss of function of the MC1R gene. The produced gel of the DNA was analyzed by checking the loss of the heterogeneity.

Results

Result 1

The table above shows the parameter results for the PCR amplification. The thermo cycler was set at the initial temperature of 940C that lasted for 5 minutes. After the initialization, the sampled DNA was subjected to 94oC for 30 seconds for denaturation. The denaturation was to ensure that the double strands of the DNA are broken down into single strands in preparation for hybridization. The denatured DNA as then subjected to 57oC for annealing of the primers to only the exon regions of the DNA.

Mutation results

Patient 1

Homozygous wild type

Parent 1 ( wild type)

Parent 2( wild type )

WT

WT

WT

WT

WT

WT

WT

WT

Patient 2

Irish mutation at Arg151Cys

Red hair

Parent 1 ( Irish mutation)

Parent 2( wild type )

Cys

Cys

Arg

Arg-Cys

Arg-Cys

Arg

Arg-Cys

Arg-Cys

Patient 3

Common mutation at Val60Leu

Fair hair not red

Parent 1 (common mutation)

Parent 2( wild type )

Leu

Leu

Val

Val-Leu

Val-Leu

Val

Val-Leu

Val-Leu

Patient 4

With C: Homozygous wild type

With t: Irish mutation at Arg151Cys , red hair

Parent 1 (Irish mutation)

Parent 2( wild type )

Arg

Cys

Arg

Arg-Arg

Arg-Cys

Arg

Arg-Arg

Arg-Cys

Parent 1 is homozygous for the wild type and has not experienced any mutation at the MC1R gene level. Both the parent 1 and 2 for the patient 1 are wild type with homogenous status. The offspring will be homogenous wild type. Patient 2 is homozygous mutant for the irish type of mutation at the Arg-Cys make him or her have red hair. The spouse, however, is homozygous wild type for the Arg type of amino acid. The red hair of patient 2 is a result of mutation which took place at the Arg-Cys amino acid region. The offspring from these couples will be heterogeneous at the Arg-Cys locus and because all of them will have allele for the wild type, none will have the red hair.

For the patient 3, there is common mutation at the Val-60-Leu and this leads to fair hair that is not red in color. The spouse is homogenous wild type for the Val amino acid. The children of these couples will also have wild type and hence will not have fair hair. However, there condition will be heterogeneous for the Val-Leu locus.

For the patient 4, parent 1 is heterozygous mutant for the irish mutation Arg-151-Cys and thus having red hair. The spouse is homozygous wild type with Arg at the MC1R gene locus. The offspring will be both heterozygous and homozygous and thus two of the offspring will have red hair while the other homozygous for the wild type and thus will not have red hair.

Gel Electrophoresis results

Discussion and Conclusion

PRC amplification has become the most common technique for the detection of the alterations at the gene level. However, the worrying thing is that the amplification of the DNA is prone to errors and these copying related errors are brought by the Taq polymerase. The above gel shows that DNA base pairs for numbers 1, 3 and 4 have the same restriction sites at the 620bp and 640bp. On the other hand, 2, 5 and 6 DNA bands also have similar base pairs at 620bp, 640bp and 1260bp and this therefore, means that they have the same restriction sites. The migration of the DNA molecules is also determined by the size apart from the negative charge which is common to all the DNA molecules. The gel results, therefore, confirm the fact that DNA bands at the 1st, 3rd and 4th position have the same size having been cut by the same restriction enzymes at the same restriction sites. Their migration on the gel will therefore be at the same level and the same applies to 2, 5 and 6 bands. The same results also shows that samples 1, 3 and 4 might have undergone gene mutation at the 1260 bp and hence remaining with only the 620bp and 640bp. In other terms, the mutational events at the MCIR gene lead to loss of certain restriction sites.

In conclusion, the polymerase chain reaction (PCR) remains the best technique for the DNA brand analysis of the mutation at various gene levels. The position of the brands at positions 2, 5 and 6 are the same a show that the DNA molecules at these positions have the same restriction sites. Similarly, the brands at the 1, 3 and 4 positions also have the same restriction sites.

Bibliography

Garibyan, L. and Avashia, N., 2013. Research techniques made simple: polymerase chain reaction (PCR). The Journal of investigative dermatology, 133(3), p.e6.

Ito, S., Nakanishi, Y., Valenzuela, R.K., Brilliant, M.H., Kolbe, L. and Wakamatsu, K., 2011. Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples: application to chemical analysis of human hair melanins. Pigment cell & melanoma research, 24(4), pp.605-613.

Lalueza-Fox, C., Römpler, H., Caramelli, D., Stäubert, C., Catalano, G., Hughes, D., Rohland, N., Pilli, E., Longo, L., Condemi, S. and De La Rasilla, M., 2007. A melanocortin 1 receptor allele suggests varying pigmentation among Neanderthals. Science, 318(5855), pp.1453-1455.

Maheaswari, R., Kshirsagar, J.T. and Lavanya, N., 2016. Polymerase chain reaction: A molecular diagnostic tool in periodontology. Journal of Indian Society of Periodontology, 20(2), p.128.

Meredith, P. and Sarna, T., 2006. The physical and chemical properties of eumelanin. Pigment Cell & Melanoma Research, 19(6), pp.572-594.

Simon, J.D., Peles, D., Wakamatsu, K. and Ito, S., 2009. Current challenges in understanding melanogenesis: bridging chemistry, biological control, morphology, and function. Pigment cell & melanoma research, 22(5), pp.563-579.