21.2 DNA sequencing Flashcards
what is DNA sequencing?
process of determining the precise order of nucleotides within a DNA molecule
what were initial methods of DNA sequencing?
bases were radioactively labelled and the gel electropheresis is carried out
what replaced radioactive labels in modern DNA sequencing?
fluorescent tags
what was the HGP?
the human genome project
massive intl project which mapped the entire human genome
what was the initial timeline for the completion of HGP?
15 years
why was the HGP achieved earlier than expected?
the automation of sequencing techniques
development of faster computers
when was the first complete human genome sequence published?
2003
what is the method by which DNA sequencing occurs by?
chain termination method
step 1 of DNA sequencing
the DNA is mixed with:
- primer
- DNA polymerase
- excess free nucleotides
- terminator bases
step 2 of DNA sequencing
mixture placed in a thermal cycler (PCR)
which causes the DNA to separate into single strands
eventually the primers bind to the strands
step 3 of DNA sequencing
DNA polymerase builds up new DNA strands by adding nucleotides with complimentary bases to the single-stranded DNA
what are attached to the terminators?
fluorescent tags
step 4 of DNA sequencing
when a terminator base binds to it’s complimentary base, the sequence is ended
this leads to many fragments being formed of different lengths
step 5 of DNA sequencing
the DNA fragments are then separated by gel electrophoresis
the fluorescent markers on the terminator bases are used to identify the final base of each fragment
how can the base sequence be identified from gel electrophoresis?
the first base (the smallest fragment) will have it’s band at the top of the plate (positive end)
by reading the preceding bands, you can identify the base sequence
how are the fluorescence of the terminators detected?
using lasers
what is the product of the chain termination method?
the order of bases shown in capillary sequencing shows the sequence of the complimentary strand of DNA which is then used to form the original DNA strand
how is a genome resembled?
they are compared with all the fragments in the database and areas of overlap are found
overview of DNA sequencing
what has replaced the use of gel plates?
a plastic side called a flow cell
what is massively parallel sequencing?
fragments of DNA are attached to a flow cell and replicated using PCR to form clusters of DNA fragments
then the coloured terminators are added
at the same time images are taken
adv of new DNA sequencing
extremely efficient
very fast
what ways can DNA sequencing be faster?
high throughput sequencing
(pyrosequencing)
entire genome sequencing
how is entire genome sequencing carried out?
genome is cut into smaller pieces using restriction endonucleases
fragments are inserted into BACs which are man made plasmids
BACs are inserted into bacteria
the bacteria divide to create colonies of cloned cells
DNA is extracted from each colony and cut up using restriction endonucleases
DNA fragments are sequenced
then DNA fragments are put together using overlapping regions to complete the entire genome
how is DNA sequencing become automised?
what is high-throughput sequencing?
faster sequencing method which is also cheaper
outline pyrosequecning
section of DNA separated into two strands
each band binds to small bead
PCR amplifies the DNA fragments on the beads
each bead is put into a well
free nucleotides are added to wells containing the beads
the wells have enzymes which emit light when when a free nucleotide binds to DNA strand
if the nucleotide is repeated, the light emitted is greater
computer analyses data to find base sequence
