DNA Sequencing/Variation Terminology
Molecular Diagnostics
(MDx) Review 565
TERM EXPLANATION
Sequencing
Dideoxy chain termination sequencing
Dideoxynucleotides (ddNTPs)
Sequencing ladder
Cycle sequencing
Pyrosequencing
Mutation
Point mutation
Determination of order of nucleotides in DNA molecule. Used to detect mutations, type
microorganisms, establish treatment for HIV & HCV.Sanger method. Most widely used method to determine DNA sequence.ddATP, ddGTP, ddCTP, or ddTTP. Chain-terminating nucleotides used in DNA sequencing.
Lack 3’-OH group required for formation of phosphodiester bonds between nucleotides.
When incorporated into growing DNA strand, extension stops.4-lane gel electrophoresis pattern obtained from dideoxy chain termination sequencing.Most commonly used method for DNA sequencing in clinical lab. Based on Sanger method.
Amplification by PCR, denaturation, annealing of primer, extension of new strand by DNA
polymerase. Extension stops when ddNTP is incorporated. ddNTPs are labeled with different
fluorescent tags, detected by capillary electrophoresis & fluorescent detector.Method to determine DNA sequence in short segments based on generation of light.
Doesn’t require electrophoresis or sequencing ladder.Permanent change in nucleotide sequence. May or may not cause disease.Mutation involving single nucleotide pair.continued...