1) It is possible to distinguish an individual from another due to the naturally occurring DNA sequence variations in a given species. A practical way of doing so is to use highly variable loci within the genome, like VNTR regions. VNTRs are classified as mini or microsatellites, depending on the length of the core sequence.
The nucleotide sequence of the repeating units of DNA tandem arrays are highly conserved among individuals within a species, but the same is not true regarding the number of repeats. Thus, the length of those tandem arrays is variable from one individual to another. This happens because of unequal crossing over events within these regions during the development of sperm and oocyte precursors and during meiosis.
Subjecting different DNA samples to Southern-blot analysis using a restriction enzyme and a short number of mini or microsatellites as probes, the result will be a pattern similar to a barcode: a complex DNA fingerprint that is, probabilistically speaking, unique for each individual.
3) In order to be detected, a DNA probe as to go through a labelling process. Depending on the technique being used, the probe may be fluorescently or radiolabelled.
There are two different processes used frequently by which a fluorescently labelled probe is created: the nick translation method and the PCR method.
The nick translation method relies on the conjugated action of two different enzymes, the DNase I and DNA Polymerase I:
- DNase I, which in the presence of Mg2+ ions becomes a single stranded endonuclease, cleaves DNA to yield 5’-phophorylated di-, tri-, and oligonucleotides (in a proportion of 60%, 25% and 15%, repectively). This enzyme shows some sequence preference when cleaving DNA: it prefers cleaving purine-pyrimide sequences and is sensitive to the minor groove structure. Despite this preferences, DNase I cuts at all four bases in heterogeneous DNA.
- Using its 5’-3’ exonucleolytic and polymerase activity, DNA Polymerase I replaces regular nucleotides with the provided dye-labelled nucleotides. Originally isolated from E. coli bacteria, this single-chain enzyme with a mass of about 109 kDa, is nowadays commercially available in a variety of formulations.
The PCR method takes advantage of the procedure invented by Saiki. In its extension step, a thermostable polymerase is used to synthesise a new DNA strand, using labelled nucleotides. There are several commercially available thermostable polymerases, the most common being Taq, Pfu and Tli (also known as Vent), named after their bacterial origins. These polymerases, which have maximal enzymatic activity at 75 to 80°C, extend complementary DNA-hybridised primers from its 3’ end using nucleotide triphophates
To radiolabel a DNA probe, three main reagents are necessary, along with the necessary buffers: an oligonucleotide, ATP containing the radioactive isotope 32P in the -phosphate position, and Polynucleotide Kinase (PNK). Kinase is the general term used to classify an enzyme that is able to transfer the -phosphate from ATP to its substrate; PNK is used to provide radiolabelled phosphate to the 5’ end of a polynucleotide chain. The mechanism by which this process occurs is the following:
PNK is a naturally occurring enzyme in the T4 bacteriophage, and commercial preparations are usually products of the cloned phage gene expressed in E. coli.
4)a) An organism that is hemizygous for a given gene contains only one copy (one allele) of that gene. It occurs as a consequence of chromosomal loss or in sex chromosome linked genes, as is the case of the X chromosome in human males, because they only carry one copy of the chromosome. Therefore, in the case of this family, the hemizygous alleles are the ones associated with the father (the 99bp and 43bp fragments)
b) The results provided by the restriction pattern give us the information that this particular mutation that affects Factor VIII in the Jackson family is associated with the absence of BclI restriction site. Therefore, it can be deduced that this is the case of a point mutation in a region of the gene that overlaps with that of the recognition sequence for the BclI restriction enzyme (since the size of the undigested fragment is equal to the sum of the size of two resulting fragments of the digestion the possibility of deletion or insertion of fragments in the sequence is discarded)
c) Analysing the restriction patterns for this family, one can deduce that the mother is a carrier of this mutation - she is heterozygous, therefore carrying a normal and a defective X chromosome. If individual 6 is a male, he is hemizygous and has inherited the normal X chromosome from his mother; if individual 6 is a female, she is homozygous and has inherited both normal X chromosomes from her parents. As such, this individual will be unaffected by this mutation and will only develop Haemophilia A due to another cause.
d) Given the fact that the mutation is known to occur in the restriction site recognised by BclI, it is possible to devise a series of PCR experiments with degenerate primers to locate the specific mutation. The primers are specifically designed to contain the sequence of the gene that presents the restriction site for BclI; each primer containing a mutation for a nucleotide in that restriction site. Knowing that the restriction site for this enzyme is T|GA TCA, it will be necessary to have seven different primers, six of them degenerate, containing a mutation in one of those six bases. Amplification of the studied DNA sequence will only be successful when the right set of primers is used. The experiment should also be carried with a positive and a negative control for the primers used.
Kogan, S.C., Doherty, M., Gitschier, J. (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A; N. Engl. J. Med. 317, 985-990.
Wang, L.K., Lima, C., Shuman, S. (2002) Struture and mechanism of T4 polynucleotide kinase: an RNA repair enzyme; EMBO Jornal 21, 3873-3880
Zhu, Z., Chao, J., Yu, H., Waggoner, A. (1994) Directly labeled DNA probes using fluorescent nucleotides with different length linkers; Nucl. Acids Research 22, 3418-3422
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