| Allele-specific oligonucleotides and dot-blot hybridization
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| This method uses a small probe to probe the blotted DNA under highly stringent conditions
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| Figure 34.12 Restriction fragment length polymorphisms (RFLP). Variations in the nucleotide sequence of DNA, either due to natural variation in individuals or as a result of a DNA mutation, can abolish the recognition sites for restriction enzymes. This means that when DNA is digested with the enzyme whose site is abolished, the size of the resulting fragments is altered. Southern blotting and probe hybridization can be used to detect this change. Results are shown for (A) homozygous normal and (B) heterozygous mutant individuals. B, BamHl restriction site. |
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| RFLPS CAN BE USED FOR DETECTING PATHOLOGIC MUTATIONS |
| A 24-year-old Afro-Caribbean woman was referred for prenatal counseling. Her younger brother had sickle-cell anemia, and she had become pregnant. Her partner was known to be a carrier of the sickle-cell mutation (sickle-cell trait) and she wanted to know if her child would develop sickle-cell anemia. |
| Since the patient is at risk of being a carrier, she opted to have chorionic villus sampling (CVS) performed to detect the presence or absence of the sickle-cell mutation in her child. Analysis of her own DNA revealed that she was a carrier, and the CVS showed that the child was also a carrier and would not develop sickle-cell anemia. |
| Comment. Occasionally, a mutation will directly abolish or create a restriction site and thus allow the use of a restriction-based method to demonstrate the presence or absence of the mutant allele. One widely examined mutation is the A > T substitution at codon 6 in the sequence for the β-globin gene responsible for sickle-cell disease. This results in a glutamine-valine (Glu-Val) mutation in the amino acid sequence β-globin and also abolishes a recognition site for MstII (CCTN(A > T)GG) in the β-globin gene. Digestion of normal human DNA with MstII and probing the Southern blot with a probe specific for the promoter of the β-globin gene yields a single band of 1.2 kb as the nearest MstII site is 1.2 kb upstream in the 5' region of the gene. The abolition of the codon 6 restriction site means that the fragment size seen when probing MstII digested DNA is now 1.4 kb, as the next MstII site is located 200 bases downstream in the intron after exon 1. Thus, patients with sickle-cell anemia will show only one band, 1.4 kb, while carriers will have 2 bands, one 1.4 kb and another, 1.2 kb, and unaffected individuals will have a single 1.2 kb band (Figs 34.12 and 34.13). |
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| The other extreme of the RFLP method is to use a small oligonucleotide probe (15-30 nucleotides) and probe the
blotted DNA under conditions of high stringency. Careful choice of temperature and solvent ensures that oligonucleotides will only hybridize to complementary sequences where there is a perfect match, down to the single nucleotide level. Mutations that result from a single nucleotide change can be detected by using oligonucleotides that recognize either the mutant or the normal allele of the gene, so-called allele-specific oligonucleotides (ASOs). ASOs can be used to screen DNA for the presence of several mutations, particularly if the template DNA is immobilized on a membrane for dot-blot analysis. Single-stranded DNA is transferred to a membrane as in the case of Southern blotting, but rather
than transferring size-fractionated digested DNA, total human genomic DNA or PCR-generated fragments are blotted onto the membrane. In this way, ASOs can be hybridized to the membrane under high-stringency conditions and autoradiography performed to determine whether the DNA contains a specific allele. The process can then be repeated for other alleles (Fig. 34.14).
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