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Walter Gilbert (1932). Marcaje radiactivo y separación de las hebras del ADN. C only: Hydrazine in 1.5 M NaCl, piperidine. C+T: Hydrazine, piperidine. G only: DMS, piperidine. A + G: DMS, formic acid, piperidine. Rompe el ADN mediante 4 reacciones químicas específicas.

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Walter gilbert 1932

C only: Hydrazine in 1.5 M NaCl, piperidine

C+T: Hydrazine, piperidine

G only: DMS, piperidine

A + G: DMS, formic acid, piperidine

Rompe el ADN mediante 4 reacciones químicas específicas














Walter gilbert 1932

+

Kary B. Mullis (1944)

Frederick Sanger (1918)

Secuenciación cíclica del ADN = Sanger + PCR


Walter gilbert 1932

TdTAMRA (600 nm)

AdR6G (570 nm)

GdROX (620 nm)

CdR110 (540 nm)

4 ddNTPs fluorescentes distintos


Walter gilbert 1932

C

R-110

A

R6G

T

TAMRA

G

ROX

Cada ddNTP emite una fluorescencia de distinto color


Walter gilbert 1932

template

Se puede hacer la reacción en un solo tubo, no en 4





Walter gilbert 1932

Electroferograma

La electroforesis capilar es mucho más rápida




Walter gilbert 1932

Electroferograma

Cada electroferograma secuencia entre 400 y 900 bases






Walter gilbert 1932

3. Pirofosfato + APS  ATP + luciferina  luz




Walter gilbert 1932

Pirograma

Reconstrucción de la secuencia (hasta 700 pb)


Walter gilbert 1932

James Watson (1928)

Pirosecuenciación de un genoma completo