Chain Termination DNA Sequencing Flashcards
What is Chain Termination DNA Sequencing?
A method of DNA sequencing meant for amplification of the DNA fragment to be sequenced by DNA polymerase and incorporation of modified nucleotides – specifically, dideoxynucleotides (ddNTPs)
Step 1
Obtain a patient sample (HAS TO BE BACTERIAL)
In DNA sequencing, we will be looking at the __________
16S rRNA gene
What is the 16S rRNA gene?
Ribosomal RNA, a small subunit of prokaryotic ribosomes.
ALL bacteria have _______
16s rRNA gene
__________ are unique to each bacterial species
Variable regions
_____________ do not change between species.
Conserved regions
Make many copies of ______________using ____________ → Necessary to copy __________
16S rRNA gene variable regions (billions of copies) , Polymerase Chain Reaction (PCR), target sequence.
First PCR requires _______, ________, _______, ________, ________, (x2)
template DNA, buffer-pH, polymerase-enzyme, dNTPs, primer (x2)
Primer
Short strand of DNA that binds to DNA surrounding target sequence. Bind to control regions to amplify variable
Primers are set down __________.
antiparallel
Polymerase will extend off the ____ end and make a copy, using _________ as template.
3’, original
The ___ and ___ indicate which part is exposed
3’, 5’
Step 2
Run PCR- Make copies of 16s rRNA gene (Variable region, flanked by conserved regions)
a) Primers bind to left and right sequence of interest to amplify variable region
b) Use primers 2x complimentary to conserved region
c) Make billions of copies
Step 3
PCR x1
a) add ddNTPs (nucleotides- sugar, phosphate and base- don’t have
3’OH) (Dideoxy- lacking 2 oxygen)
b) DNA polymerase pulls bases onto sequence, some fall off, some create sequence
