Quick Review Cards for Medical Laboratory Science

Sanger Dideoxy Chain Termination Method

Molecular Diagnostics

(MDx) Review 567

Principle

Components

Procedure

Modification of DNA replication that incorporates labeled dideoxynucleotides (ddNTPs, chain terminating

nucleotides) in reaction mixture. Similar to dNTPs except lack 3’ OH needed for phosphodiester bond

formation. When incorporated into growing DNA chain, elongation is terminated.

Template, primer, DNA polymerase, dNTPs (dATP, dCTP, dGTP, dTTP), ddNTPs (ddATP, ddCTP, ddGTP, ddTTP)

1. DNA template amplified.


2. 4 reaction tubes containing:
   a. template
   b. primer
   c. DNA polymerase
   d. all 4 dNTPs
   e. only 1 ddNTP (ddATP, ddCTP, ddGTP, or ddTTP)


3. Amplicon denatured.


4. Primer hybridizes to target.


5. DNA polymerase extends primer, occasionally incorporating ddNTP that stops further extension.
   Resulting fragments are of various lengths. All fragments in tube end with same labeled ddNTP.


6. Products electrophoresed in 4 separate lanes labeled A, C, G, T, corresponding with the ddNTP in tube.
   Fragments separate according to size.


7. Gel dried & exposed to x-ray film, producing sequencing ladder. Band furthest from origin is smallest,
   fastest migrating fragment & ends in the 1st nucleotide in the sequence, e.g., if band furthest from
   origin is in lane A (lane from tube that contained ddATP), then 1st nucleotide in sequence is A. Ladder
   is read from bottom to top to determine entire nucleotide sequence.

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