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1977
Two different sequencing techniques published
Maxam-Gilbert Sequencing
Chemical degredation
Partial digest, rarely used
Sanger-Coulson Method
Chain termination
enzymes build pieces of DNA and then a gel is ran
First step of M-G
End is labeled with radioactive phosphate using kinase
What happens after you label the DNA in MG
Heat strands to seperate and run a gel to get the strand that you want and extract with a scapel
What happens in MG once you have your strand?
Seperate into 4 tubes
Hydrazine Tube
Only modifies C+T
Hydrazine and 5M NaCl tube
Modifies C
Formic Acid tube
modifies A and G
Dimethyl sulfate tube
modifies G
What to do once bases are modified
Piperidine is added to break the strands where the bases are modified
Why is the chemical degredation only done partially?
So different bases are modified on each peice of DNA and each strand is cut differently
This means that all of the bases cna be determined in the gel
What to do once bonds are broken?
Run gel and sequence
How to develop MG gel electrophoresis
Press the gel onto an X-Ray film paper
Why is M-G not used often?
toxic chemicals
piperdine explodes
tedious
expensive
When is MG still used over SC
When sequencing areas with lots of secondary structure
Dideoxy nucleotides
Have no 3' hydroxyl group, stop DNA strand from expanding when added to a strand
Components of Sanger-Colson Sequencing
Polymerase: Klenow, Sequase, KlenTaw
Primer for one strand
Nucleotides (95 % dntp and 5 % ddntp)
Detective Agent
Buffer
Start of S-C sequencing
Primer binds to denatured template
What happens after the primer binds?
The Polymerase elongates the strand until a ddntp is added
What happens after a ddntp is added?
Elongation stops, this creates many strands of every possible length
How many reactions are used in older S-C sequencing
4, one for each ddntp nucleotide
What to do after elongation stops in S-C sequencing?
Run each reaction side by side on a gel
Detecting Sequences in S-C
Was radioactive S 35
Now we use fluoresence attached to the ddntp molecules
What does using flouresence allow us to do to modify the gel electrophoresis?
We can now run all four reactions in one lane, a laser scans each of the bands as they pass sequentially
Capillary Electrophoresis
Used to sequence S-C sequences now
Uses polyacrylamide instead of agarose
Uses one lane
Two kinds of Next Gen Sequencing
Reversible Terminator
Pyrosequencing
Reversible Terminator Method
Each nucleotide has a fluorescent blocking group in the hydroxyl spot that is released and creates a certain color light whenever it is added to the DNA chain
Lights are detected using lasers
Pyrosequencing
Adding a dntp to a DNA strand releases a pyrophosphate
This group then reacts with a modified ADP with a phosphate and a sulfate group using the enzyme Sulfurylase to make ATP
ATP serves as a coenzyme to activate luciferase which reacts with luciferin to create light
Pyrosequencing: Wrong vs right nucletoide added
Wrong: degrades
Right: makes a flash of light detected by the sensor, the machine will tell you which base it added to make the light
Sequencing Multiple Strands
The genome is fragmented, amplified, and immobilized by a glass slide/universal adapter or metallic bead with avidin and biotin with large numbers of primers of know sequences on the surfaces
Two Genome Sequencing Approaches
Shotgun
Hieracrchical
Shotgun Approach
DNA is broken into random pieces, cloned into vectors, and plugged into a computer to sequence and determine an order
Shotgun limitations
Only good for small genomes
Repeated elements split between clones are deleted
Hierarchical
Cloned large genomes are split and connected by vectors and overlap is found
They are then split into smaller pieces in a different vector and overlap is found again
This process repeats
Why is a Hierarchical Approach better for Eukaroyotes?
They have larger genomes with more repeating elements
How are gaps from the Hierarchical Approach bridged traditionaly?
probes are made to look for a specific clone
If two clones bind the same probe, then they likely overlap/are contigs
4 Newer ways to overlap clones
Chromosome Walking
Clone Fingerpirnting
Repetitive DNA PCR
Sequenced Tagging Sites
Chromosome Walking
A clone is selected and tested to see what it will hybridize
Clone Fingerprinting
Chromosome walking with multiple clones at once
Repetitive DNA PCR
Search clone banks for clones with the same distance between repetitive elements
Sequence Tagged Sites
Info from previousaly sequenced DNA is used to make primers for PCR to id the clones
3 Mapping Methods
RFLP's
Microsatellites
SNPs
RFLPs
presence or absence of restriction sites leads to different size fragments in gel electrophoresis
Used for gene fingerprinting
STR/VNTR
Different numbers of tandem repeating elements can be detected by PCR + Gel electrophoresis
SNP
differences in single nucleotides, many detection methods
3 Main SNP detection Methods
1. Use probe that can attach to one variant but not other
2. Same but with primer
3. Invader Assay
Invader Assay
If probe matches then light appears
If it does not no light appears because probe cannot anneal to sequence
Physical Mapping
Use radiation hyprids (hamster cells with one human chromosome)
Add FISH probes made from cDNA to find location of genes