How is RFLP analysis used to detect genetic abnormalities?

Found this for you: look hereL http://www.ndsu.edu/pubweb/~mcclean/plsc431/markers/marker1.htm

basically: If a particular RFLP is usually associated with a particular genetic disease, then the presence or absence of that RFLP can be used to counsel people about their risk of developing or transmitting the disease. The assumption is that the gene they are really interested in is located so close to the RFLP that the presence of the RFLP can serve as a surrogate for the disease gene itself. But people wanting to be tested cannot simply walk in off the street. Because of crossing over, a particular RFLP   might be associated with the mutant gene in some people, with its healthy allele in others. Thus it is essential to examine not only the patient but as many members of the patient's family as possible.

The most useful probes for such analysis bind to a unique sequence of DNA; that is, a sequence occurring at only one place in the genome. Often this DNA is of unknown, if any, function. This can actually be helpful as this DNA has been freer to mutate without harm to the owner. The probe will hybridize (bind to) different lengths of digested DNA in different people depending on where the enzyme cutting sites are that each person has inherited. Thus a large variety of alleles (polymorphisms) may be present in the population. Some people will be homozygous and reveal a single band; others (e.g., all the family members shown below) will be heterozygous with each allele producing its band.

RFLP and Genetic Screening

Restriction fragment length polymorphisms (RFLP) have become key elements in systems to diagnose diseases. Let's look at how these experiments are performed. First, as with any genetic comparison we need variable phenotypes and some way to detect them. Human diseases can be detected clinically. Sickle cell anemia patients can be detected by the shape of their blood cells, and hemophiliacs can be identified by the inability of their blood to coagulate properly. But we now have the capability to detect in utero indivi duals that will express these traits.

Two tools are needed to perform these DNA type diagnoses. First we need a restriction enzyme that will cut DNA in a manner that is informative for the disease in question, and second we need a nucleic acid probe that will detect that region. Not all restriction enzyme sites will reside in the same position in all individuals. Thus if we cut DNA with a restriction enzyme we may not obtain the same size DNA fragments. For example:



Sample A:  |  1 kb  |         4 kb         |
           _____________________________ ____
Probe:     XXXXXXXXXXXXX

Sample A:  |             5 kb              |
           _____________________________ ____
Probe:     XXXXXXXXXXXXX


Note: The symbol "|" denotes the restriction enzyme cleavage site.

The procedure that is used to analyze the DNA is Southern hybridization. The two DNA samples are cut with the appropriate enzyme and are hybridized with the correct probe. The probe hybridized to the 1 and 4 kb fragments in sample A and the 5 kb fragment in sample B. This is an example were a restriction site has been gained in sample A (or lost in sample B) by mutation.


        Sample A   Sample B
        -------------------

5.0 kb               ___
4.0 kb    ___
 


1.0 kb    ___

        -------------------

Now let's look at a second example.


Sample A:  |     3 kb     |     3 kb       |
           _____________________________ ____      
Probe      XXXXXXX

Sample A:  |   2 kb   |       4 kb         |
           _____________________________ ____      
Probe      XXXXXXX

For this example, the Southern Hybridization would look like this.



        Sample A   Sample B
        -------------------
 
3.0 kb    ___

2.0 kb               ___


        -------------------
In this example a 3 kb fragment is recognized in sample A and a 2 kb fragment is recognized in sample B. These two examples demonstrate a powerful technique that is becoming widely used and accepted: restriction fragment length polymorphism (or RFLP). These two examples demonstrate how this name is derived. All the samples we are looking at are examples of restriction enzyme fragments. In each example we see a polymorphism between the size of the fragments that are recognized by the probe.
It is important to remember that any RFLP is unique to a specific restriction enzyme and nucleic acid probe.

RFLP Screening for Human Diseases

Let's look at specific examples. The first RFLP example will detect an individual with sickle cell anemia. Remember that sickle cell is the result of a change in the #6 amino acid of the ß- globin chain of hemoglobin. Specifically glutamic acid is converted to valine. This results from a change in the nucleotide A to T. This change eliminates a site recognized by the restriction enzyme DdeI.
Restriction enzyme: DdeI (recognition sequence: 5'-GTNAG-3')
Probe: fragment of ß-globin coding sequence


                                   Site
                                Eliminated
Normal ß-Globin  |   175 bp |    201 bp     |
  Allele         ____________________________   
Probe            XXXXXXXXXXXXXXXXXXX


Sickled ß-Globin |            376 bp        |
  Allele         ____________________________      
Probe            XXXXXXXXXXXXXXXXXXX


 
         Normal   Sickled
        ß-Globin  ß-Globin
        ------------------


376 bp               ___

201 bp     ___
175 bp     ___

        ------------------
Each of the two marker patterns (376 bp vs. 201 + 175 bp) can be considered alleles. These markers can also be used to describe a biochemical phenotype.
The second example is for a DNA markers for human X-linked disease hemophilia A. This disease affects 1/10,000 males. The affected gene produces Factor VIII a component of the blood coagulation pathway. This his gene is mutated in affected patients.

For Hemophilia A two alleles exist:

1.2 kb fragment = hemophilia allele
0.9 kb fragment = normal allele
Restriction enzyme: BclI
Probe: Fragment from Factor VIII coding region
 
Normal Allele       |    0.9 kb    | 0.3 kb |
                    _________________________
Probe               XXXXXXXXXXXXX

Hemophilia Allele   |         1.2 kb        |
                    _________________________
Probe               XXXXXXXXXXXXX

Representation of an autoradiogram for hemophilia individuals


        Normal Normal  Carrier Affected Affected
         Male  Female  Female   Female    Male
        ----------------------------------------

1.2 kb                  ___      ___2x     ___

0.9 kb   ___    ___2x   ___

        ----------------------------------------
2x means the intensity of the band is twice that of the other bands.
Why is the female band twice as intense? Because they contain two X chromosomes, therefore twice the amount of DNA.