DNA SEQUENCING TECHNIQUES Flashcards
Elucidates the order or
sequence of nucleotides in
a DNA chain
DNA Sequencing
Most definitive molecular
method to identify genetic
lesions
DNA Sequencing (STILL)
Requires double- or singlestranded DNA samples with one end that is radioactively labeled (_______________)
template
- Templates are aliquoted to 4
tubes - Each tube is treated with a
different chemical (see table) - Reduction using _______________
- Evaporation of _______________
piperidine (for both???)
- Drying and resuspension in
formamide - _______________ gel separation
- Drying of gel and exposure to
light - Analysis of data
polyacrylamide
Sanger
- _____________________________
- Modification of the DNA
replication process - Detects products using a
radioactive or fluorescentlabeled nucleotide
dideoxy chain termination sequencing
- 1:1 mixture of _______________ into 4
separate reaction tubes with
sequencing enzymes and buffer for
polymerase activity - All dNTPs and ¼ ddNTPs into each
tube - DNA polymerase is added and
reaction runs for _______________
template:primer
about 20 mins
- Addition of a stop buffer (EDTA,
_______________, loading dye) - Loading into denaturing
polyacrylamide gel - Dry the gel and visualize the
fragments - Analysis of ___________________
stop buffer
sequencing buffer
stinct colors (_______________) of
fluorescence emission pertains to the
appropriate nucleotide and is the
basis of the read instead of the lane
peak wavelenghts
Four different fluorescent dyes
are attached to four separate
aliquots of the _____________
primer
Fluorescent dyes are covalently
attached to each _______________
instead of the _______________
ddNTP
primer
Product fragments are labeled
at the _______________
3’end
Electrophoresis
Detectors convert
fluorescence to an
electrical signal
(____________________)
electropherogram
PYROSequencing
Relies on ______________ when
nucleotides are added to a growing strand of DNA
luminescence
bisulfite dna sequencing
________________-specific sequencing
methylation
Fragments are denatured by heat
and exposed to bisulfite solution
(___________________, NaOH, and
___________________)
___________________= converted to uracil
___________________= unchanged
sodium bisulfate
sodium hydroxide hydroquione
unmethylated cytosine
methylated cytosine
Next gen sequencing: Illumina
GOALS:???
✓ Make sequencing the entire genome accessible
✓ Make genomic studies a part of routine in research
and the clinic
Modification of ________________
sequencing to detect methylated
nucleotides and is less concerned
with generating a new sequence
chain termination
REACTION MIX: Single-stranded DNA template, sequencing primer,
______________ and ______________, ______________, ______________
sulfurase, luciferase and adenosine 5 phosphosulfate, luciferin, polymerase
Preparation of Ladder
After the sequencing reaction, excess terminators are removed
Clean with: ______________
or by ______________
columns or heads
ethanol precipitation
