DNA Sequencing Flashcards
DNA Sequencing Methods
Direct
Pyrosequencing
Bisulfite
NGS
Direct (2)
Manual
Automated
Direct - Manual (2)
Chemical (Maxam-Gilbert)
Sanger
Direct - Automated (2)
Dye Primer
Dye Terminator
NGS (2)
Illumina*
MinION*
Most definitive molecular
method to identify genetic
lesions
DNA Sequencing
Elucidates the order or
sequence of nucleotides in
a DNA chain
DNA Sequencing
Maxam-gilbert
•______ Sequencing
• Requires_________ samples with one end that is______
Chemical double- or single-
stranded DNA
radioactively labeled
(template)
• Chemical Sequencing
• Requires double- or single-
stranded DNA samples with one
end that is radioactively labeled
(template)
Maxam-gilbert
Maxam-gilbert
1. Templates are aliquoted to____
2. Each tube is treated with a
different chemical
3. Reduction using_____
4. Evaporation of piperidine
5. Drying and resuspension in
_____
6. _____separation
7. Drying of gel and exposure to
____
8. Analysis of data
4 tubes
piperidine
formamide
Polyacrylamide gel
light
Madam Gilbert
• For sequencing, the labeled fragment, or template is aliquotted into_____
• Each aliquot is treated with a different chemical with or w/o high salt
• Upon addition of a strong reducing agent (e.g._____), the single-stranded DNA will break at specific nucleotides
• After reactions, piperidine is evaporated, and contents of each tube are dried and resuspended in_____ for gel loading
• The fragments are then separate dby size on a denaturing____
* The denaturing conditions ( formamide, urea, and heat ) PREVENT the single strands of DNA from hydrogen bonding with one another or folding up so that they migrate through the gel STRICTLY ACCORIDNG TO THEIR____.
Migration speed is important because single-base resolution
is required to interpret the sequence properly.
• After electrophoresis
• Gel apparatus is disassembled
• Gel is removed to a sheet of filter paper; then dried on a gel dryer
• Dried gel is exposed to light-sensitive film
• Wet gels can be exposed directly
four tubes
10% piperidine
formamide
polyacrylamide gel
SIZE
4 Base Modifiers used in Maxam-Gilbert Sequencing
Dimethylsulphate
Formic acid
Hydrazine
Hydrazine + salt
G
Dimethylsulphate
Methylated G at 4C
G+ A
Formic acid
Protonates proteins at 5C
T+ C
Hydrazine
Splits pyrimidine rings at 8C
C
Hydrazine + salt
Splits only C rings at 8 C
• Impractical for high-throughput
sequencing of long fragments
• HAZARDOUS REAGENTS!
Maxam-gilbert
Madam Gilbert reagent
• - toxic & flammable
Piperidine
Toxic and combustible
Irritant!
Flammable
Madam gilbert base modifiers
• Detects products using a
radioactive or fluorescent-
labeled nucleotide
Sanger
• Modification of the DNA
replication process
Sanger
• Dideoxy chain termination
Sanger
Products of a Maxam-Gilbert sequencing reaction.
The gel is read from the_____-_____.
The____ of the fragments gives the order of the nucleotides.
The nucleotides are inferred from the lane in which each band appears.
A or T is indicated by bands that appear in the G + A lane or C
+ T lane, respectively, but not in the G lane or the C lane.
bottom to the top
size
An alternative detection strategy is to incorporate 32p- or 35S-labeled deoxynucleotides in the nucleotide sequencing reaction mix
A.k.a INTERNAL LABELING
Sanger
SANGER
- 1:1 mixture of____ and _____ into 4 separate reaction tubes with sequencing enzymes and buffer for polymerase activity
- All _____ and 1/4_____into each tube (different ddNTP in each of the four tubes)
a. Ratio of ______is critical for generation of a readable sequence
b._________ → polymerization will terminate too frequently early along the template
c._________ → infrequent or no termination will occur
3._______ is added and reaction runs for 20 minutes
template and primer
All dNTPs and ¼ ddNTPs
ddNTPs/dNTPs
TOO HIGH CONC.
TOO LOW CONC.
DNA polymerase
- Addition of a stop buffer 20mM (2)
a.______ to chelate cations and stop enzyme activity
b._____ to denature the products of the synthesis reaction
c. Gel loading dyes (2)
EDTA, Formamide loading dye
EDTA
Formamide
bromophenol blue/xylene cyanol)
- Loading into______
a. Products of each of the four sequencing reactions are loaded into adjacent lanes → labeled A, C, G, or T
6._____ the gel and visualize the fragments (expose to x-ray film)
a. Fragment patterns are visualized by the signal on the 32p-labeled primer or nucleotide
b. All fragments from a given tube will end in the same ddNTP
- Analysis of sequencing ladder
a. Four-lane gel electrophoresis pattern of the products of the four sequencing reactions is called a_____
b. Read to deduce the DNA sequence
denaturing polyacrylamide gel
Dry
sequencing ladder
Sanger
1.) 1:1 mixture of ______into 4 separate reaction tubes with
_____ and_____for polymerase activity
2.) All ____ and _____into each tube
3.) DNA polymerase is added and
reaction runs for____
4.) Addition of a stop buffer (3)
5.) Loading into_____
6.) Dry the gel and visualize the
fragments
7.) Analysis of_____
template:primer ; sequencing enzymes and buffer
dNTPs and ¼ ddNTPs
~20 min
EDTA, formamide, loading dye
denaturing polyacrylamide gel
sequencing ladder
Larger bands can be compressed at
times, limiting the length of the
sequence and is thus harder to read
Improvements to fluorescent dye
technology and the introduction of
thermal cyclers have led to
automation of sequencing
Sanger
An improvement to Sanger Sequencing
AUTOMATED Sequencing
Uses double-stranded templates and cycle sequencing that doesn’t require sequential addition of reagents to start and stop the reaction
Automated sequencing
Distinct colors (peak wavelengths) of
fluorescence emission pertains to the appropriate nucleotide and is the basis of the read instead of the lane
Automated sequencing
Madam Gilbert
___%- polyacrylamide gels ran with _____/_____loading dye
6-20%
bromophenol blue/xylene cyanol
Sanger
Label can be in the ____end of the primer or specific nucleotides have incorporated radioisotopes or fluorescent molecules
5’
Sanger
- lacks the 3’ hydroxyl group needed to form a phosphodiester bond with the phosphate group of another nucleotide.
ddNTP
Sanger
________ may be added to the sequencing reaction to promote equal incorporation of dNTPs for uniform band intensities and to increase relative incorporation of ddNTPs
Manganese
Sanger
______ and_____may also be added to avoid secondary structures in the synthesized fragments
Deaza-dGP and deoxyinosine triphosphate
AUTOMATED SEQUENCING
Dye primer
• Four different fluorescent dyes
are attached to four separate
aliquots of the primer
• Products are labeled in the____
end with the dye color associated to the ddNTP at the end of the fragments
5’
AUTOMATED SEQUENCING
DYE terminator
• Fluorescent dyes are covalently
attached to each_____
instead of the primer
• All four sequencing reactions
are performed in the____
• Product fragments are labeled
at the____ end
ddNTPs
same tube
3’
AUTOMATED SEQUENCING
• Four different fluorescent dyes
are attached to four separate
aliquots of the primer
• Products are labeled in the 5’
end with the dye color
associated to the ddNTP at the
end of the fragments
Dye primer
AUTOMATED SEQUENCING
• Fluorescent dyes are covalently
attached to each ddNTPs
instead of the primer
• All four sequencing reactions
are performed in the same
tube
• Product fragments are labeled
at the 3’ en
DYE terminator
AUTOMATED
After the sequencing reaction, excess terminators are removed
Clean with: columns/ beads or by ethanol precipitation
Denaturation of the fragments
Preparation of Ladder
AUTOMATED
Ran on the same lane, avoiding lane-to-lane migration variations
Migrating fragments pass a laser beam and then a detector
Detectors convert fluorescence to an electrical signal (electropherogram)
Electrophoresis
AUTOMATED
Dye Blobs bright flashes of fluorescence due to failure to clean the ladder
Poor starting material leads to poor quality sequence
Sequence interpretation
Reading the sequence
Most useful for short to moderate sequence analysis or single nucleotide polymorphisms (SNP) and typing rather than for generating new sequences
PYROSequencing
Relies on luminescence when
nucleotides are added to a growing
strand of DNA
PYROSequencing
Determines a DNA sequence without having to make a sequence ladder
PYROSequencing
REACTION MIX: Single-stranded DNA template, sequencing primer,
sulfurylase and luciferase, APS, luciferin
PYROSequencing
Bisulfite DNA sequencing
• Important as____ of DNA affects cell differentiation and is implicated in several diseases such as cancer
• Modification of chain termination sequencing to detect ______and is less concerned with generating a new sequence
•______ of genomic DNA is cut with restriction enzymes to facilitate denaturation and the fragments are resolved in AGE
• Fragments are denatured by____ and exposed to ______for_____
• Cytosines are deaminated = converted to uracil 5-methyl cytosines. = unchanged
methylation
methylated nucleotides
2 to 4 ug
heat; bisulfite solution (sodium bisulfate, NaOH, and hydroquinone)
16 to 20 hours
• Methylation-specific sequencing
Bisulfite DNA sequencing
Bisulfite sequencing
Unmethylated C =
Methylated C =
converted to uracil
unchanged
Designed to sequence large numbers of templates simultaneously, yielding hundreds of thousands of sequences in just a few hours
Next gen sequencing: Illumina
GOALS:
✔ Make sequencing the entire genome accessible
✔ Make genomic studies a part of routine in research and the clinic
Next gen sequencing: Illumina
