pliz help!!!!! - midicloria

[ArchivalUser]

i really need to know how to read southerblot , westerblot, analysis with agarose gel electroforesis....

[ArchivalUser]

it is basically a giant matching game.....

First you have to understand the concept of Electrophoresis. It uses electricity to seperate either DNA, RNA, or proteins based on size of molecule. Therefore, when you run a certain solution the different size proteins will separate. If you then add a radiolucent tag to them you can visualize at what points on the gell the certain structures are. So in the case of HIV for example you get different proteins due to the nucleocapside, envelope..and other proteins etc and this will stay consistant for any HIV protein. So now if you run a known sample of HIV and then run a patients blood if the bands match up you may suspect the patient is positive for HIV. (That was an example of a western blot) Its fairly straight forward.

The fairly complex issue comes into play with northern and southern blot. Remember this is checking for the presence of certain genes. Therefore, recessive and dominant disorders are important in determing if the person will present with the phenotype or not. Lets take an example of a Recessive disorder, sickle cell.(this is a simplification but expresses the concept) First you run a gel that has two controls(one control is positve for the mutation and the other control is normal gene) On that same gel you run the two parents DNA and their 3 kid's DNA. Now you analyze the bands formed....

First the Parents: Both parents show a band at the level of the positive control(has sickle cell) and one band at the normal control(does not have sickle cell). This tells us that each parent has one gene that is positive and one negative for sickle cell. In a reccessive disorder such as sickle cell we call this sickle cell trait. The parents are normal phenotypically but are heterozygous genomically and therefore can pass the defective gene onto their kids.

Second we test the oldest KId: He shows the same pattern as the parents(one band at the positive trait and one at the negative) therefore he is heterozygous but does not present with the disorder.(as we know in a recessive disorder you need two defective genes to present).

Third we check the well of the middle kid: the band pattern only shows a very thick dark band at the level that matches with the positive control for sickle cell. This kid therefore has no good genes and has inherited both mutated genes from his parents. The reason the band is thicker and darker is because unlike in the older kids serum there is a split between where the DNA goes in the gel 1/2 to the positive and 1/2 to the negative in this kids well all the DNA goes to the same location on the gel and appears darker and bigger. Therefore as we can see this kid has sickle cell.

Lastly we check the youngest kids well: He has one big dark band at the level of the negative control band. We can conclude that this kid has inherited both GOOD genes from his parents and therefore is not even a carrier. the band is darker and thicker for the same reason as explained above.

HOPE THIS HELPED.....Remember to keep in mind the inheritance pattern of disease in question. Because unlike a recessive disorder if it is a dominant disorder you only need one gene to be mutated to present phenotypically and therefore if there is a split as we saw in the oldest kid he would still present becasue he has atleast one bad gene.

GL

[ArchivalUser]

thank you very much i understood you are very nice

take care and continue doing good things...

bye