A PCR restriction enzyme analysis
takes advantage of the occurrence of
approximately 40 thalassemia mutations that either introduce or remove a
restriction endonuclease site. The PCR amplified target sequence is digested
using restriction enzymes (enzymes that cleave DNA at particular nucleotide
sequences) and the pattern of fragmentation on an agarose gel defines the pres-
ence or absence of a particular mutation.
Radio-labeled single stranded DNA oligonucleotide probes are allowed to
anneal with the target strand in an
oligonucleotide hybridization analyses.
Typically, the target DNA is fixed onto nitrocellulose or nylon membrane. The
probe forms stable duplexes with target sequences from the heterogenous mix-
ture of many sequences in genomic DNA and is detected by autoradiography
using an x-ray film. In populations that have predominantly one common
mutation, a very successful and efficient hybridization analysis is the
allele-specific oligonucleotide (ASO) assay.
This requires that the sequence of
the common mutation is known. The amplified genomic DNA is transferred
onto a nylon membrane and probed using an allele-specific oligonucleotide,
that is, one that is complementary to the sequence of the DNA that contains
the mutation. The membrane is probed both with oligonucleotides having the
mutant and the correct DNA sequence of the P-globin gene. The genotype of
the DNA is determined by the presence or the absence of the hybridization sig-
nal. Recently, microchips, which are an array of oligonucleotides immobilized
on a glass plate, have been used to detect thalassemias. Fluorescent labeled DNA
from an individual is hybridized onto this microchip and the reaction is mon-
itored with a fluorescent microscope. This technique allows the study of a spe-
cific mutation using several hundred oligonucleotides simultaneously. It is
highly sensitive with a low background; however, the resolving power is low.
Using one or a more of the DNA analysis tools described above, effective
prenatal detection of various mutations in the P-globin gene cluster has been
successfully achieved. Although gene therapy for genetic diseases is in its
infancy, hopefully the future will bring effective treatment options for patients
affected with thalassemia.
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