DNA Sequencing

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49 Terms

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1977

Two different sequencing techniques published

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Maxam-Gilbert Sequencing

Chemical degredation

Partial digest, rarely used

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Sanger-Coulson Method

Chain termination

enzymes build pieces of DNA and then a gel is ran

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First step of M-G

End is labeled with radioactive phosphate using kinase

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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

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What happens in MG once you have your strand?

Seperate into 4 tubes

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Hydrazine Tube

Only modifies C+T

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Hydrazine and 5M NaCl tube

Modifies C

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Formic Acid tube

modifies A and G

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Dimethyl sulfate tube

modifies G

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What to do once bases are modified

Piperidine is added to break the strands where the bases are modified

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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

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What to do once bonds are broken?

Run gel and sequence

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How to develop MG gel electrophoresis

Press the gel onto an X-Ray film paper

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Why is M-G not used often?

toxic chemicals

piperdine explodes

tedious

expensive

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When is MG still used over SC

When sequencing areas with lots of secondary structure

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Dideoxy nucleotides

Have no 3' hydroxyl group, stop DNA strand from expanding when added to a strand

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Components of Sanger-Colson Sequencing

Polymerase: Klenow, Sequase, KlenTaw

Primer for one strand

Nucleotides (95 % dntp and 5 % ddntp)

Detective Agent

Buffer

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Start of S-C sequencing

Primer binds to denatured template

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What happens after the primer binds?

The Polymerase elongates the strand until a ddntp is added

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What happens after a ddntp is added?

Elongation stops, this creates many strands of every possible length

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How many reactions are used in older S-C sequencing

4, one for each ddntp nucleotide

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What to do after elongation stops in S-C sequencing?

Run each reaction side by side on a gel

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Detecting Sequences in S-C

Was radioactive S 35

Now we use fluoresence attached to the ddntp molecules

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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

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Capillary Electrophoresis

Used to sequence S-C sequences now

Uses polyacrylamide instead of agarose

Uses one lane

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Two kinds of Next Gen Sequencing

Reversible Terminator

Pyrosequencing

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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

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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

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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

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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

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Two Genome Sequencing Approaches

Shotgun

Hieracrchical

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Shotgun Approach

DNA is broken into random pieces, cloned into vectors, and plugged into a computer to sequence and determine an order

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Shotgun limitations

Only good for small genomes

Repeated elements split between clones are deleted

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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

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Why is a Hierarchical Approach better for Eukaroyotes?

They have larger genomes with more repeating elements

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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

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4 Newer ways to overlap clones

Chromosome Walking

Clone Fingerpirnting

Repetitive DNA PCR

Sequenced Tagging Sites

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Chromosome Walking

A clone is selected and tested to see what it will hybridize

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Clone Fingerprinting

Chromosome walking with multiple clones at once

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Repetitive DNA PCR

Search clone banks for clones with the same distance between repetitive elements

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Sequence Tagged Sites

Info from previousaly sequenced DNA is used to make primers for PCR to id the clones

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3 Mapping Methods

RFLP's

Microsatellites

SNPs

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RFLPs

presence or absence of restriction sites leads to different size fragments in gel electrophoresis

Used for gene fingerprinting

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STR/VNTR

Different numbers of tandem repeating elements can be detected by PCR + Gel electrophoresis

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SNP

differences in single nucleotides, many detection methods

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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

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Invader Assay

If probe matches then light appears

If it does not no light appears because probe cannot anneal to sequence

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Physical Mapping

Use radiation hyprids (hamster cells with one human chromosome)

Add FISH probes made from cDNA to find location of genes