Molecular Biology: DNA sequencing
Molecular Biology:
DNA sequencing
Author: Prof Marinda Oosthuizen
Licensed under a Creative Commons Attribution license.
DEFINITIONS
Annealing: Binding of an oligonucleotide primer to a DNA or RNA template.
Codon: A triplet of nucleotides in a messenger RNA molecule coding for a single amino acid.
Contig: an aligned contiguous section of sequence comprising two or more DNA sequences joined
together by virtue of matching bases.
Deoxynucleoside triphosphates (dNTPs): The basic building blocks of DNA, consisting of a
nitrogenous base (adenine, guanine, thymine, or cytosine), a phosphate molecule, and a sugar
molecule (deoxyribose).
Dideoxynucleoside triphosphates (ddNTPs): Modified dNTPs that lack a 3' hydroxyl (3'-OH) group,
and therefore terminate strand synthesis when incorporated into a growing DNA chain.
DNA polymerase: An enzyme that synthesizes DNA on a DNA or RNA template.
DNA sequencing: The experimental process of determining the exact order of nucleotide bases in a
DNA molecule.
Dye-labelled terminators: ddNTPs that are labelled with fluorescent groups.
Fluorophore: A molecule that can be excited by light to emit fluorescence.
Genetic code: The sequence of nucleotides, coded in triplets (codons) along the mRNA, that
specifies the sequence of amino acids in the translated protein. The DNA sequence of a gene can be
used to predict the mRNA sequence, and the genetic code can in turn be used to predict the amino
acid sequence.
Melting temperature (Tm): The temperature at which two strands of a double-stranded DNA
molecule detach due to breakage of the hydrogen bonds between them.
Open reading frame: A series of codons each of which encodes an amino acid, uninterrupted by a
stop codon (TAA, TAG or TGA). All ORFs have the potential to encode a protein or polypeptide
although many may not actually do so.
1|P a g e
Molecular Biology: DNA sequencing
Polyacrylamide: A material used to make gels for separation of mixtures of macromolecules (such
as proteins, small DNA or RNA molecules of up to 1000 nucleotides) by electrophoresis.
Polyacrylamide is used in DNA sequencing gels to separate the sequencing ladder.
Primer: A short oligonucleotide that, when annealed to its complementary sequence in a singlestranded DNA template molecule, provides a start point for synthesis of a new DNA strand.
Reading frame: One of three possible ways of translating a nucleotide sequence into codons.
Subcloning: The transfer of a cloned fragment of DNA, or a part thereof, from one vector to another.
Template DNA: The DNA molecule that is copied during a strand synthesis reaction. In a sequencing
reaction, strand synthesis is catalysed by a DNA polymerase.
Thermostable DNA polymerase: A DNA polymerase that is relatively unaffected by high
temperatures. Taq polymerase is the most commonly used thermostable DNA polymease and was
isolated from Thermus aquaticus, a bacterium found in hotsprings in Yellowstone National Park in the
United States of America.
2|P a g e