Example: The normal factor V gene has two restriction sites for the enzyme Mn11, one of which is lost if there is a mutation in the factor V gene. This results in the production of different-sized products when DNA from normal and affected individuals is amplified by polymerase chain reaction (PCR) then cut with Mn11.The different products are visualized on gel electrophoresis.

2. Oligonucleotide probe analysis is used when the point mutation producing the abnormal gene does not alter any known restriction site.

Two oligonucleotides 18 and 20 bases long are synthesized, having at their centers the single base by which the normal and mutant genes differ.

Each oligonucleotide hybridizes strongly to the corresponding (normal) gene but weakly to the gene that does not share the exact sequence.

Thus after PCR amplification of target DNA, the normal and mutant genes can be distinguished on the basis of the strength of the hybridization with the two oligonucleotide probes.

3. Mutations that affect the length of DNA (Eg: deletion or expansion) can also be detected by PCR analysis.

Example: In the fragile X syndrome, amplification of the DNA by primers that flank the region affected by trinucleotide repeats, generate products of different sizes, when DNA from normal carrier males and affected individuals is compared.

Indirect Gene Diagnosis:

Gene Tracking:

In many genetic diseases, the mutant gene and its normal counterpart have not yet been identified or sequenced, and thus direct gene diagnosis cannot be used.

It is therefore necessary to employ gene tracking, which determines whether a given family member or fetus inherited the same relevant chromosomal region(s) as a previously affected family member.

This technique requires that chromosomes carrying the normal or mutant genes in heterozygotes be distinguishable.

To accomplish this, advantage is taken of naturally occurring variations in DNA sequences in the vicinity of (and linked to) the mutant gene.

Such variations may result from differences in specific nucleotides (site of polymorphisms) or from differences in the number of nucleotide repeats (length polymorphism).

Site polymorphisms, also called restriction fragment length polymorphisms, results from DNA polymorphisms that give rise to fragments of different lengths in Southern blot analysis.

In cystic fibrosis, for example, heterozygous parents and children have two bands derived from the normal and the affected chromosome.

In contrast, an affected (homozygous) individual reveals a single band derived from two identical chromosomes carrying the mutant gene.

Length polymorphisms result from difference between the number of repeats of short sequences of non-coding DNA.

These may be detected by PCR analysis of the DNA because the product size depends on the number of nucleotide repeats.

Linkage analysis has proved useful in antenatal detection of several genetic disorders, such as cystic fibrosis, Huntington disease, polycystic kidney disease, fragile X syndrome, and Duchenne muscular dystrophy.

It has certain limitations, however, as follows:

- For prenatal diagnosis, several affected and unaffected family members must be available for testing.

- 2 Key family members (Eg: parents, siblings) must be heterozygous for the polymorphism (i.e. the normal chromosome and that carrying the mutant gene must be distinguishable).

- Recombination between homologous chromosomes during gametogenesis may lead to loss of linkage between the DNA polymorphism and the mutant gene.