Lecture 6 - Profiling and Sequencing Flashcards
what are the differences between profiling and sequencing (3)
profiling
1. looks at allele level differences
2. info on familial relationships
3. used in forensic science
sequencing
1. looks at base-level sequences
2. info on proteins expressed
3. biomedical analysis
4. can also trace mutations
what is the one similarity between profiling and sequencing
they both rely on polymerase reactions
what are the three sequencing methods we have looked at
sanger sequencing
NGS = next generation sequencing
nanopore sequencing
what are ddNTPs
dideoxynucleoside 5’-triphosphates
how do dNTPs and ddNTPs differ in sanger sequencing
dNTPs have and OH group on carbon 3 that ddNTPs don’t have
dNTPs are the monomers used in DNA synthesis and ddNTPs terminate the DNA synthesis
the lack of the OH group gives no space for the next base to add on
what is sanger sequencing
how does it differ from PCR
a method used to determine the sequence of nucleotide bases in a a DNA molecule
specific use of ddNTPs in reaction mix are used to terminate the polymerisations so no further bases are added
when a ddNTPs adds to the strand the strand stops being added to - this is random we don’t control it
primers are still used to signal the beginning of where polymerase should add
PCR uses primers for this termination
how can the different fragment lengths made in sanger sequencing be visualised
on a gel using electrophoresis
e.g a PAGE gel
what does a 15mer stand for
15 base pairs on length
how can a complete sanger sequence set be obtained for a sample
repeat the process 4 times
each using a different ddNTP (one with ddATP then ddCTP then ddTTP then ddGTP)
so you will see where the terminations happen for each
in sanger sequencing what do the position of the bands seen in electrophoresis show
the position of the the base complimentary to the one used in the ddNTP
e.g if ddATP was used the bands show the positions of T in the template DNA sequence and A in the newly synthesised sequence
what can be obtained from a sanger sequence
looking at the gel from the electrophoresis can give the complementary DNA sequence and then we can work out the template strand sequence from this
the complimentary runs 3’-5’ (top to bottom of the gel)
top of gel = smaller fragments
the original sequence is the complementary one to what the gels tells us
what was originally used for sanger sequences to be visualised on gels in electrophoresis
why didn’t this work
what was used instead
using Phosphorus 32 labelled ddNTPS (radiaoactive phosphorus)
interpretation could be hard as smudging was seen between bands and each base being the same colour made it confusing
labelled each ddNTP with a fluorescent dye instead - each base having a different colour dye = quicker and easier to see = observe under UV light
what can be done to measure many samples at once in sanger sequencing
use automated capillary systems for electrophoresis rather than a gel
what is next generation sequencing
how is it better than sanger sequencing (5 reasons)
new modern technology allowing rapid and parallel sequencing of DNA or RNA
1 = it can sequence millions of fragments in one run whereas sanger only does 1 fragment
2 = is can read much longer DNA sequences
3 = much quicker
4 = cheaper than sanger
5 = automated
how does NGS work (brief) (5 steps)
1 = fragment genomic DNA - vibrate using ultra sound e.g
2 = isolate the individual fragments onto separate beads/surfaces
3 = use PCR to amplify the different fragments
4 = add ddNTPs one at a time (do this 4 times - one for A, T, C, G)
5 = sequence fragments
