Manipulating Genomes Flashcards
What is DNA sequencing
The process of determining the precise order of nucleotides within a DNA molecule
What is sanger sequencing
Different sized fragments of partially copied DNA enable the sequence of DNA to be determined
What are key concepts of sanger sequencing
-Chain terminating nucleotides are being incorporated
-Dideoxynucleotides are used (they lack an-OH group on carbon 3)
-Often radioactive primers are used to help locate the fragments on the gel as they will show up when x-rayed the method can be used to sequence genes >1Kbp and gave scientists the potential to describe the whole genome
What are the ingredients of sanger sequencing
• DNA polymerase enzyme
• A primer- short piece of single-stranded DNA that binds to the template DNA and acts as a “starter” for the polymerase
• The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• The template DNA to be sequenced
• Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labelled radioactively
What is the method of sanger sequencing
• “Ingredients” are added to a tube
• The mixture is first heated to separate the strands
• Then cooled so that the primer can bind to the single-stranded template.
• DNA polymerase synthesises new DNA starting from the primer.
• DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.
• This process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction.
• That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA.
• The ends of the fragments will be labelled with radioactive primer
