about PCR

The gene MC1R is responsible for directing the production of the melanocortin 1 receptor protein. For healthy pigmentation, the receptor is crucial. The receptor is mostly found on the surface of melanocytes, which create melanin (Wallis, 2015). Humans differ in their skin and hair color due to polymorphisms in the MC1R gene. The protein melanocortin 1 receptor's capacity to induce the formation of eumelanin is diminished as a result of the polymorphisms. Even though the MC1R gene is essential for human skin pigmentation, other genes also play an important part in the determination of the skin and hair colouring (Wallis, 2015). It plays a significant role in determining sun sensitivity. The study of MC1R is a major genetic risk factor for melanoma and non-melanoma skin cancer. Studies on MC1R may give insights on skin lightening.

Polymerase Chain Reaction commonly known as PCR is a technique that is used to amplify a copy or copies of a segment of DNA to produce millions of DNA sequence in molecular biology (Abraham, 2012). The reaction targets specific regions of DNA. The technique is based on the ability of DNA polymerase to produce or synthesize new strands of DNA strands that are complementary to the template strands that are offered. PCR analysis enables scientists to perform DNA analysis on tiny samples such as the skin cells (Nagui, Mahmoud, Abdel, Hassieb, & Rashed, 2015). The technique can be used to detect infectious disease or in the field of molecular biology for forensic analysis (Disotell, 2017).

Gel electrophoresis is a laboratory technique that is used to separate the mixtures of RNA, DNA or the proteins per their molecular size (Berg, 2012). Molecules are separated by an electric field using a gel with small spores (Berg, 2012). All of the DNA molecules have the same charge per mass thus gel electrophoresis of the DNA fragments makes it possible to separate them based on their size. The analysis is necessary for DNA testing (Berg, 2012). The technique is used in the DNA analysis because of the ability to visualize the fragments.

Methods

Practical 1: PCR design on the human MC1R gene

The identification of the MC1R mutation consequences in human beings was made using the available websites to get the mutation phenotypes that cause the loss of function for MC1R protein. The NCBI web page was used for the identification of MC1R genomic sequence.

The primer3web Web page was used for the designing of a primer pair for the amplification of TRANSLATED EXON of the human MC1R gene with the target annealing temperatures of 57°C. The primer pair amplified all the translated exon of MC1R gene.

Practical 2: Buccal Swab, DNA isolation, and PCR

A BuccalAmp DNA Extraction Kit was used to collect the swab samples for the PCR analysis. It was rotated inside the cheek of a person for approximately 20 times to collect the sample and heated for 1 minute at 65°C and 2 mins in 98°C in a QuickExtract DNA extraction solution before the sample is ready for analysis.

Practical 3: PCR Analysis and Gene Clean

PCR products were cleaned using QIAquick PCR purification kit. A simple, fast bind-was-elute procedure and an elution volume of 30-50 µl is used for DNA purification. Result A pH indicator is used to determine the optimal pH for the DNA binding to the spin columns

Preparation of 1% agarose and running the gel

1% agarose TAE buffer is boiled in a microwave oven in the flask. SYBR safe is added before pouring. (5ul in 50ml.5ul of the PCR product is checked before in 1% of the agarose gel and a solution of 5 µl DNA and 1 µl loading dye 6X. 100 mV gel is run for 15 minutes with 3 µl of the molecular weight.

PCR Product Purification

5 volumes of buffer PB were added to 1 volume of PCR sample and mixed. Mineral oil or kerosene is not removed. When pH indicator I was added to the PB buffer, the colour of the mixture was checked to be yellow. It was ensured by adding 10ul of 3M sodium acetate, pH 5 and mixed if the colour was found to be violet or orange.

QIAquick spin column was provided in a 2ml collection tube.

The sample was applied to the Q1Aquick column and centrifuged for 30-60 seconds to bind the DNA.

The QIAquick column was placed in the same tube again to discard the flow-through.

0.75 ml Buffer PE was added to the QIAquick column and centrifuged for 30-60seconds for washing.

The flow-through was then discarded, and the QIAquick column placed back in a similar tube centrifuge for an additional 60 seconds.

The QIAquick column was placed in a clean 1.5ml microcentrifuge tube.

Elution of the DNA was done by adding 50ul Buffer EB to the centre of QIAquick membrane and the column centrifuged for 1 min.

30ul elution Buffer was added to the centre of QIAquick membrane to increase the concentration of DNA and then centrifuged for 1 minute.

1 volume of loading dye was added to 5 volumes of purified DNA for gel analysis.

The clean PCR product was stored at -20°C.

Practical 4: Restriction Enzyme Digestion of PCR Product

The requirements were Clean PCR product: 10 μl, Enzyme Buffer (10X): 3 μl, Enzyme XXX: 1 μl, Water: 16 μl

They were digested overnight, and agarose gel analysis conducted the following day. The image of the v schematic representation of the gel for the digestion was uploaded.

The gel was run at 100 mV for 15 mins with 3ul of the same molecular weight marker.

The mutation was identified as suggested by the targeted digestion enzyme.

The DNA translation tool (http://translate-protein.com/) was used

The restriction enzyme map tool (http://www.restrictionmapper.org/) was used for the identification

The enzyme that can be used for the detection of loss of function mutation and the preparation of the digestion of PCR product with the enzyme

Sequencing of the PCR product

Two rounds of sequencing were done per product. One was done using forward primer and the other one using the reverse primer. A clean PCR product 4 μl was prepared in a labelled tube for each sequencing reaction.

Practical 5: Bioinformatics analyses of the MC1R gene amplicon

The web page http://web.expasy.org/translate/ was used to translate DNA into protein. The NCBI website was used for the alignment of the two sequences. The sequence that was given by the demonstrators was compared to the reference sequence. The mutations that changed the coding sequence were identified and the amino acids compared to the changes to identify the mutations that lead to the phenotypic differences.

Results

Practical 1: The PCR parameters that were determined based on the primer pair.



Practical 3

The following are the results of the gene clean that was stored for subsequent digestion and sequencing



Practical 4

The PCR digestion and sequencing produced the following results.



Practical 5

The results of sequence analysis of MC1R gene were identified. The sequence was identified if the patient was homozygous wildtype, heterozygous or homozygous mutant. The resulting phenotype of each patient was also identified.

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



Discussion

Patient 1

The spouse of patient 1 is a homozygous wild-type. The crossing of the two parents will produce progeny with the same genotype.

Patient 2

Parent 1 is a homozygous mutant for the Irish mutation Arg-51-Cys that causes red hair. On the other hand, the second parent is a homozygous wild-type for the amino acid Arg. Children of these parents will be heterozygous for the particular locus. They will bear the Irish mutation and the amino acid. However, all the children will have the wild-type allele thus no red hair caused by the mutation.

Patient 3

Parent 1 is a homozygous mutant for the common mutation Val60Leu that is for fair hair not red. Parent 2 is a homozygous wild-type for the amino acid Val. The progeny of the parents will, therefore, be a crossing of the two thus resulting in being heterozygous for the particular locus. The children will all have the wild-type allele thus not expressing the common mutation.

Patient 4

For patient 4, parent 1 has the Irish mutation ARG151Cys for red hair. On the contrary, parent 2 is a homozygous wild-type with the amino acid Arg. The crossing between the two parents will produce all progenies without an expression of red hair. They will all be carrying the alleles of wild-type and two will be taking the red hair. Two of the children will have different features concerning the red hair.

Conclusion

The preparation and the clean-up of the DNA for sequencing is a crucial step as it determines the quality and the precision of the results. The different scenarios that were identified based on the spouse genotype express the genetic crossing of the different genes and mutations. The expression of a mutation in a progeny is only possible when the alleles of the mutation are present in the offspring. The offspring with both the wild-type alleles do not express the mutations as shown in the analysis results.



References

Abraham, J. M. (2012). Detection and Isolation of Differentially Expressed Genes by PCR. PCR in Bioanalysis, 239-244. doi:10.1385/0-89603-497-6:239

Berg, H. (2012). Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis - Valuable Tool for Genotyping and Genetic Fingerprinting. Gel Electrophoresis - Principles and Basics. doi:10.5772/37724

Disotell, T. R. (2017). Polymerase Chain Reaction (PCR), Quantitative/Real-Time PCR (qPCR), Reverse Transcriptase PCR (RT-PCR). The International Encyclopedia of Primatology, 1-2. doi:10.1002/9781119179313.wbprim0483

Nagui, N. A., Mahmoud, S. B., Abdel Hay, R. M., Hassieb, M. M., & Rashed, L. A. (2015). Assessment of gene expression levels of proopiomelanocortin (POMC) and melanocortin-1 receptor (MC1R) in vitiligo. Australasian Journal of Dermatology, 58(2), e36-e39. doi:10.1111/ajd.12408

Wallis, Y. (2015). Denaturing Gradient Gel Electrophoresis. PCR Mutation Detection Protocols, 125-135. doi:10.1385/1-59259-273-2:125