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9. Disease Analysis with Molecular Techniques.pdf

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Biology (Sci)
BIOL 202
Tamara Western

Disease Analysis with Molecular Techniques: Targeting diseases based on DNA is like finding a needle in a haystack or a nucleotide in an entire genome  Sickle cell anemia patients have irregular shaped red blood cells, and also have irregular hemaglobin which can cause health problems o Since the difference in these diseases is the hemaglobin protein, patients can be tested by having their hemaglobin proteins run out on a starch gel to separate by size  The Hb mutation is the cause of a single nucleotide change o A wild type individual will have one “normal” band on the gel, while a recessive mutant for HbS (sickle cell) will have one “mutant” band  Heterozygotes, which contain copies for both the mutant and wild types genes, will have two bands, one normal and one mutant  Heterozygotes for the sickle cell trait can have their blood viewed under the microscope; under normal oxygen conditions, their RBCs look normal and flow properly o Only in low oxygen conditions do the sickle cell traits start to appear, and the blood begins to have flow problems; this is due to a conformational change dependent on the [O ] 2 Dr. Allison in 1949 obtained some results from African populations which showed natural selection at work on humans for the sickle cell trait  She found that in two tribes in Africa, Luo and Suba, of different ethnic origins, they had almost the same frequency of HbS, about 25% o However, the Suba tribe and the Kikuyu tribes, which were of different ethnic origin, had very different frequencies; Suba about 25% and Kikuyu close to 0%  There was no correlation with the blood type of the populations, so what gave such high diversity between individuals? o It turns out the tribes that had high frequencies of HbS lives near the lake, while the low frequency tribe lived inland  Also living near the lake are mosquitos, particularly the Anopheles category which are hosts for Plasmodium falciparum which causes malaria o Upon biting someone, the mosquitoes can develop the bacteria, incubate it, and upon biting someone else, transfer it to them  These malarial parasites once in humans live within RBCs; giemsa staining of RBCs under the microscope stain RBCs in light pink leaving the bacteria in purple because of their DNA (RBCs normally don’t have any DNA) Survival analysis of malaria showed interesting results based on analysis of the genotypes of children  While HbSS (homozygous recessive) patients dies of sickle cell anemia early in life, HbAA (wild type) were also dying a lot because of malaria  However, the heterozygotes showed the highest success because the one allele of Hbs conferred malaria resistance, while functional HbA could still be made o This brings up the question: should we cure malaria? There’s no right answer  Even more interestingly, the distribution of malaria in Africa is centered around the central portion of the continent o The frequency of HbS almost mimics this pattern, again being centered in the central portion of Africa, which further demonstrates the high correlation between HbS and malaria protection The evolution of HbS detection has moved from proteins to DNA, at which point we noticed that the disease is caused by a single point mutation  Turning an A  T causes the change from HbA to HbS  It also changes the restriction site for a restriction enzyme, which allows us to tell the difference between the HbS gene and the HbA gene on a gel o Again, this is using the Southern blot technique which begins by restriction fragment digestion  The mutant restriction site will no longer be cut by the restriction enzyme because it is no longer recognized; mutation can also introduce new cut sites, both of which are useful in disease detection RFLPs, or restriction fragment length polymorphisms, allow us to tell the genotype difference between individuals; different genotypes will produce different fragment lengths depending on whether or not they gained/loss a restriction enzyme cut site  In this example, the mutation introduces a new target site for the enzyme o Two heterozygotes are crossed and the resulting offspring are tested based on RFLPs  The A/A type will have no restriction sites and therefore will only have 1 band  The a/a type will have 2 genes both with the new restriction site, and thus both copies of the DNA will be cut, producing 2 bands  The A/a type, however, will have both an uncut and a cut fragment, and they will therefore have 3 bands on the gel  Other types of mutations can also be detected using RFLPs: o An insertion or deletion mutation into an RFLP will produce longer (insertion) or shorter (deletion) RFLPs than the wild type  I.e. they will migrate less (insertion) or more (deletion) in the gel than the RFLP o A deletion may also remove a restriction site entirely, thus creating a larger fragment which will move less in the gel than the WT o Alternatively, an insertion mutation may also introduce a site into the RFLP, causing it to be cut into smaller fragments; both of which will move farther in the gel than the WT  In combination with pedigrees, RFLPs can become useful tools in determining the genotypes of a whole ancestral line o Taking again the sickle cell anemia example, we can see that all heterozygotes will have 3 bands when cut with an MstII restriction enzyme because the HbS mutation introduces a new MstII site in the Hb gene  Attaching a hybridization probe to the RFLPs knowing the Hb sequence of course, will allow staining specifically of the Hb genes an no other genomic content o Similarly, HbAA will only have 1 “heavy” band and the HbSS will have 2 “light” bands which were cut by the MstII  Now, we only need to use RFLPs, and no longer need to take blood samples to carry out the starch gel protein analysis There are, however, some disadvantages to using RFLPs:  Finding RFLP is often accomplished by “trial and error” before sequencing of the genome took place o Finding the right restriction enzyme was therefore a pain  Most of them are di-allelic, and thus not very informative on large a population o Either then enzyme will cut it, or it won’t which will only inform about two alleles (e.g. HbS vs. HbA)  RFLP analysis required a large amount of DNA, about 6 micrograms, which is a lot  It is time consuming (DNA isolation, DNA digestion, gel electrophoresis, Southern blot) o Making the probes in itself is time consuming This brought on the polymerase chain reaction, PCR, which has revolutionized the way we isolate and purify DNA to be used in labs  It is a technique that allows the amplification (copying) of a selected DNA sequence in a genome a millionfold or more o In this process, primers are added to a sample of DNA taken from a cell, and allowed to replicate with a Taq polymerase which can function in high temperatures o Eventually, the pieces with excess ends of DNA will be outnumbered by the amounts of DNA containing only desired sequence o After about 25 cycles of replication, the ampli6ication of what you started with has gone to about 10 , or a millionfold  It was devised by Kary Mullis (Nobel price 1993), and it has changed the way we are doing molecular biology Applying PCR to the sickle cell anemia case, we can still use the MstII to cut the RFLPs, but the isolation process is so much easier  All you need is 10 or so nanograms of the DNA containing the desired fragments; carrying out PCR will amplify this amount a millionfold  With so much of only the DNA you want, the bands will be visible without even having to carry out probing or Southern Blot o The undesired fragments will not get amplified and you won’t
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