Determination of the number of oligo:nucleotide repeat units

摘要

Determination of the number of repeat units of a motif having <= 3 of the four nucleotide bases in a repeating sequence (pref. a microsatellite) which is included in a nucleic acid sequence, coupled to a solid support, comprises: (a) hybridisation of a nucleotide primer with a complementary sequence to that situated 5' of the microsatellite; (b) addn. of (i) 2'-deoxynucleotide 5'-triphosphates (dNTPs), (ii) 2',3'-dideoxynucleotide triphosphate (ddNTP) (opt. labelled) and (iii) 3'-substd. dNTPs opt. labelled on the substit.; which respectively (i) allow normal elongation of the chain; (ii) irreversibly block chain elongation or (iii) reversibly block chain elongation; (c) adding nucleic acid polymerase under conditions allowing any of NTPs (i)-(iii) to be incorporated; (d) allowing 3' chemical or esterase hydrolysis of the chain; (e) sepn. of the supernatant of the nucleic acid sequence obtd. and measurement of labels released in the supernatant; (f) repeating steps (b)-(e); (g) counting the number of repetitions after comparing the presence or absence of label between each repeat.