DNA sequencing and analysis Flashcards
What does DNA sequencing mean?
Process of determining precise order of nucleotides within DNA molecule.
What is Sanger sequencing?
Use of chain-terminating nucleotides during DNA replication
Terminating bases prevent DNA polymerase from making new DNA strand
1st step of Capillary method:
DNA fragments mixed with primers, DNA polymerase, excess of normal nucleotides + terminator bases
2nd step of Capillary method
Mixture placed in thermal cycler
96 = double strands separate into single strands
50 = primers anneal into DNA strands
60 = DNA polymerase starts to build up new DNA strands by adding nucleotides with complimentary base to single strand DNA template.
What happens when a terminator base is added in the sequence?
Terminator base added = synthesis of DNA is terminated –> no more bases can be added
Results in DNA fragments of different lengths.
After many cycles –> all possible DNA chains will be produced.
How are the DNA fragments separated in the capillary method?
Similar to gel electrophoresis
Fluorescent markers in terminator bases used to identify final base of each fragment
Laser detects different colours thus order of sequence.
How are the fragments compared?
Data of sequencing process fed into computer –> reassembles genomes by comparing all fragments + finding areas of overlap between them.
Can identify genes that code fro specific characteristic.
What is next generation sequencing?
Technology =faster/automated
Sequencing takes place on plastic slide (flow cell)
uses computer technology = cost fallen +more time efficient
