cytology-cytogenetics: Procedure For DNA Fingerprinting

Procedure for DNA fingerprinting involves following steps:

    1. Performing a Southern Blot
     2. Making a Radioactive Probe
     3. Creating a Hybridization Reaction
     4. VNTRs

Southern Blot

The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. Performing a Southern Blot involves:

1. Isolating the DNA in question from the rest of the cellular material in the nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA,or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.

2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.

3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.

4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.

5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.

In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question (see next topics for more information). If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds [red] will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.

Making a Radioactive Probe

1.Obtain some DNA polymerase [pink]. Put the DNA to be made radioactive (radiolabeled) into a tube.



2.Introduce nicks, or horizontal breaks along a strand, into the DNA you want to radiolabel. At the same time, add individual nucleotides to the nicked DNA, one of which,*C [light blue], is radioactive.

3.Add the DNA polymerase [pink] to the tube with the nicked DNA and the individual nucleotides. The DNA polymerase will become immediately attracted to the nicks in the DNA and attempt to repair the DNA, starting from the 5' end and moving toward the 3' end.

4.The DNA polymerase [pink] begins repairing the nicked DNA. It destroys all the existing bonds in front of it and places the new nucleotides, gathered from the individual nucleotides mixed in the tube, behind it. Whenever a G base is read in the lower strand, a radioactive *C [light blue] base is placed in the new strand. In this fashion, the nicked strand, as it is repaired by the DNA polymerase, is made radioactive by the inclusion of radioactive *C bases.



5.The nicked DNA is then heated, splitting the two strands of DNA apart. This creates single-stranded radioactive and non-radioactive pieces. The radioactive DNA, now called a probe [light blue], is ready for use.
- Creating a Hybridization Reaction
  
  Creating a Hybridization Reaction
  
  1. Hybridization is the coming together, or binding, of two genetic sequences. The binding occurs because of the hydrogen bonds [pink] between base pairs. Between a A base and a T base, there are two hydrogen bonds; between a C base and a G base, there are three hydrogen bonds.
  
  2. When making use of hybridization in the laboratory, DNA must first be denatured, usually by using heat or chemicals. Denaturing is a process by which the hydrogen bonds of the original double-stranded DNA are broken, leaving a single strand of DNA whose bases are available for hydrogen bonding.
  
  3. Once the DNA has been denatured, a single-stranded radioactive probe [light blue] can be used to see if the denatured DNA contains a sequence similar to that on the probe. The denatured DNA is put into a plastic bag along with the probe and some saline liquid; the bag is then shaken to allow sloshing. If the probe finds a fit, it will bind to the DNA.
  
  4. The fit of the probe to the DNA does not have to be exact. Sequences of varying homology can stick to the DNA even if the fit is poor; the poorer the fit, the fewer the hydrogen bonds between the probe [light blue] and the denatured DNA. The ability of low-homology probes to still bind to DNA can be manipulated through varying the temperature of the hybridization reaction environment, or by varying the amount of salt in the sloshing mixture.
  - VNTRs(Variable Number Tandem Repeats)
    - Every strand of DNA has pieces that contain genetic information which informs an organism's development (exons) and pieces that, apparently, supply no relevant genetic information at all (introns). Although the introns may seem useless, it has been found that they contain repeated sequences of base pairs. These sequences, called Variable Number Tandem Repeats (VNTRs), can contain anywhere from twenty to one hundred base pairs.
    - Every human being has some VNTRs. To determine if a person has a particular VNTR, a Southern Blot is performed, and then the Southern Blot is probed, through a hybridization reaction, with a radioactive version of the VNTR in question. The pattern which results from this process is what is often referred to as a DNA fingerprint.
    - A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have. Shown below are the VNTR patterns for Mrs. Nguyen [blue], Mr. Nguyen [yellow], and their four children: D1 (the Nguyens' biological daughter), D2 (Mr. Nguyen's step-daughter, child of Mrs. Nguyen and her former husband [red]), S1 (the Nguyens' biological son), and S2 (the Nguyens' adopted son, not biologically related [his parents are light and dark green]).
    - Because VNTR patterns are inherited genetically, a given person's VNTR pattern is more or less unique. The more VNTR probes used to analyze a person's VNTR pattern, the more distinctive and individualized that pattern, or DNA fingerprint, will be.
    Source:http://protist.biology.washington.edu/fingerprint/dnaintro.html