#ddntps

[er-cryptid]

Chain Termination PCR

-- also called Sanger sequencing

-- used to determine the nucleotide sequence of DNA

-- manual or automated

-- PCR occurs first

-- DNA is denatured to break the double-strand into two single strands

-- primers anneal to each strand

-- they extend to create two double strands

-- in Sanger sequencing / chain termination, ddNTPs are added along with dNTPs

-- ddNTPs can be added randomly as the chain extends

-- they stop the chain

-- this is because they can’t form phosphodiester bonds like dNTPs can

-- next, the products are separated by size

-- this is done with gel electrophoresis

-- DNA polymerase works in the 5′ to 3′ direction

-- each terminal ddNTP will correspond to a specific nucleotide in the original sequence