Info

1.35 kb

1.15 kb

Direction of electrophoresis

Southern blot of restriction digest

Figure 1.17 Use of restriction enzymes to genotype the sequence variant responsible for Hb S. The T to A substitution at the DNA level results in loss of the restriction enzyme recognition site for MstII. Restriction enzymes are highly specific such that they cut particular nucleic acid sequences: in the case of MstII, 5'-CCTNAGG-3' where N is any nucleotide. In the presence of the A to T substitution the recognition site is lost (CCTGTGG). Restriction enzymes are derived from bacteria, for example MstII originates from Microcoleus species. Southern blotting using a probe hybridizing to DNA sequence 5' to HBB gene results in a Southern blot where particular sizes of fragments allow individuals with sickle cell trait or sickle cell disease to be identified versus unaffected individuals.

Target sequence

Forward primer

Reverse primer

Denature

Primers anneal

Primer extension (DNA synthesis)

First PCR cycle

Denature

Primers anneal

Primer extension (DNA synthesis)

Second PCR cycle

Denature Primers anneal Primer extension (DNA synthesis)

Third PCR cycle

20-30 cycles

Figure 1.18 Polymerase chain reaction (PCR) amplification. Exponential amplification of a target DNA sequence can be achieved by using a pair of forward and reverse primers complementary to the region of interest, a heat-resistant DNA polymerase enzyme such as Taq polymerase, dNTPs, and specific buffer including divalent and monovalent cations (magnesium and potassium). Repeated cycles of denaturation, primer annealing, and template extension allow rapid and exponential amplification; typically 20-30 cycles are performed. * denotes newly synthesized strand; ** denotes a new strand of delimited length (by locations of primers). Redrawn from Young (2005), by permission of Oxford University Press.

Escherichia coli DNA polymerase I enzyme and variants detected by restriction enzyme digestion using Ddel (Saiki et al. 1985). This paper demonstrated how exponential amplification of DNA was achieved (some 220 000-fold increase after 20 cycles of PCR) and that 200 times less

DNA was required: 20 nanograms versus two micrograms for Southern blotting.

As an alternative to restriction enzyme digestion, allele-specific detection was developed in which an oligonucleotide probe with or without the sequence

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