Sequencing Flashcards
what is sanger sequencuing
sanger chain termination sequencing uses ddntps that lack the free oh group needed for polymerase to add new nucleotides. this causes the chain to terminate at a certain legnth, this can be combined with gel electrophoresis and colored flouro-ntps to determine the order
what is required for sanger sequencing
ddntps
regular dntps
polymerase
primers
what is the limit of sanger sequencing
500-800 bases
what are some types of next generation sequencing
pyrosequencing and nanopore
what is the constructive terms used for sequencing and what order do they go in
contigs
scaffolds
chromosomes
what are the two forms or orginisational structures in sequencing
random or overlapping
what are coverage and confidence
coverage represents the number of sequences of a partical part and confidence is the number that concur
what are some benefits of pyrosequecing
no electrophoresis
is massivley parralell
more rapid that chain termination
what are the steps of pyrosequencing
dna is fragmented
adaptors are ligated to ends
biotin bount to 3’ end
biotin is attached to a bead
strand duplicated using pcr
primers attach to 3’ end
nucleotides of a certain type used then washed away
what is the reaction that causes the light of pyrosequencing
atp sulpherase plus aps and pyrophosphate reacts with luciferin to create light
what is gap filling hybridisation screening in general sequencing
if a sequence hybridises to others, it cann be inferred that they sit sex to each other
larger vectors can construct the sequence using walking hybridisation
what are some benefits of nanopore sequencing
requires less setup than pyro
uses long reads
more portable
what is nanopore sequencing
nanopore sequencing uses a membrane pore used for biofilms, a motor protein, and some current. as the strand passes through the bases obstruct the current and give a measurement which is converted into the bases
