formation of phospodiester bond

nucleophilic attack

3' OH of attached sugar to alpha phosphate of incoming nucleotide

formation of ester bond in dna synthesis

ddNTP

chemically modified nucleotides

has H instead of OH at 3'

used in Sanger method

polyacrylamide gel vs. agarose gel

polyacrylamide can seperate single stranded DNA molecules differing in length by only one nucleotide

important for Sanger method of genome sequencing

old school Sanger method--can you mix the different bases together?

no different experiments for each of the four dd bases

run each experiment on different gel lane

each ddNTP has a different fluorescent label

allows all reactions to be carried out in a single tube or on one lane of a gel

advantages of capillary gel electrophoresis vs. polyacrylamide gel electrophoresis

allows much higher electric fields and thus faster rates

uses liquid polymers which may be easily flushed

sub-microliter volumes

can run hundreds of experiments in parallel and read all with single laser beam

steps of making a genomic library

1. extract and purify the DNA

2. break it into pieces

3. cloning--insertion into a plasmid

4. transformation

5.grow bacteria on plate

6. colony picking

mechanical shearing

requires large amts of DNA

DNA fragment size is highly variable

DNA flowed through tube with narrow contraction; liquid accelerates from narrow part; drag forces due to the acceleration rip the DNA molecules apart

ways to break the DNA into smaller fragments

mechanical shearing

restriction enzyme digestion

sonication (uses ultrasound)

two essential features of plasmids

(for cloning DNA)

1. gene for antibiotic resistance

so you can tell which bacteria have been transformed

2. lac z gene

so you can tell which plasmids are recombinant

plasmid

rings of self replicating DNA

ampicillin

antibiotic

bacteria can have gene for resistance for this antibiotic

x-gal

in gene cloning, used to tell whether a cell expresses the enzyme β-galactosidase or not, which is encoded by the lacz gene

β-galactosidase + x-gal = blue colony

electroporation

applying an electric field so as to change the permeability of the bacterial membrane in order to indroduce recombinant plasmids for cloning

can all bases be added at the same time with FLX 454?

no.

bases need to be added sequentially.

using plasmid as a cloning vector

max insert size

example

10-20 kb

pBR322 and pUC18

pyrosequencing

advantages

disadvantages

advantages: does not require clones or electrophoresis

disadvantages:shorter reads

sequencing methods that use emulsion PCR

FLX 454

and

SOLiD

adapters

used in

FLX 454

SOLid

Solexa

added to both ends of fragments

used for purification, amplification, and sequencing steps

in FLX 454, amplitude of peak is proportional to

# of bases added in one base cycle

(in other words, # of same base added at a time is given by the height of the signal)

FLX 454 vs SOLid

both amplify DNA via emulstion PCR

FLX 454 -- wells;

sequencing method: pyrosequencing

SOLid -- optic fiber slide;

sequencing method: ligation

does SOLid employ sequencing by synthesis?

No.

primer is oriented 3'-5'.

anneal and ligate.

how are bases tagged in SOLid?

di-nucleotide color coding

color coded according to two known bases

probe has eight bases

two are known

last three bases cleaved after ligation, leaving five: two known, three unknown

why are ligation rounds made for five different primer rounds in SOLid?

to allow for double base interrogation

how is DNA amplified in Solexa?

bridge amplification

what is the sequencing method in Solexa?

modern Sanger method

with reversible terminators

comparison of sequencing methods in terms of read length

order of methods from highest to lowers read length

large   1. Sanger

medium 2. FLX 454

small 3. Solexa / Ilumina and SOLid

summary comparison of sequencing methods in terms of base pairs per run

large Solexa and SOLid

medium  FLX 454

smaller Sanger

Sanger:

read length

bp per run

read length: 900 bp

bp per run: 96 kb

FLX 454

read length

bp per run

read length: 200-300 bp

bp per run: 80-120 Mb

Solexa

read length

Bp per run

read length: 30-40 bp

Bp per run: 1 Gb

SOLid

read length

Bp per run

read length: 35 bp

Bp per run: 1-3 Gb

Steps to shotgun approach to sequencing

1. isolate and sheer DNA

2. create library

3. sequence individual clones

4. assemble sequences into contigs

5. fill gaps between contigs

sequence gaps vs. physical gaps

(in shotgun sequencing)

sequence gaps--gaps that can be closed by further sequencing of clones already present in the library

physical gaps--stretches of sequence not present in clone library; need to return to genomic DNA and either make a second library or PCR genomic DNA with gap ends

how to fill "sequence gaps"?

can be closed by further sequencing clones already present in the library--design new primers to fill the gap

How to fill the "physical gaps"

shotun method

two methods

1. A. make a second library in different vector

B. probe second library with the ends of contigs where gaps are

C. identify clones hybridized with 2 or more oligonucleotides

D. sequence the new clones

2. A. PCR genomic DNA with remaining pairs of oligonucleotides (from ends of contigs where gaps are)

B. ID pairs that yield PCR products

C. Sequencing PCR products fills the gaps

problem of shotgun approach

two solution?

tandemly repeated DNA

and genome-wide repeats

solution: use positional markers

clone fingerprinting techniques

clone fingerprinting techniques

way to do what?

four examples

way to ID overlap in sequences obtained

1. restriction fingerprint (shared restriction frags)

2.repetitive DNA fingerprint (blotting with genome wide reps)

3.repetitive DNA PCR (shared PCR products e.g. Alu seqs)

4. STS content mapping (PCR looking for shared STS--sequence tagged site)

types of positional markers

and what are they used for?

tags in DNA sequence used to help id sequence overlaps in shotgun method

restriction fragments sites

Alu seqs

known genome wide repeats

STS (sequence tagged site)

Hierarchical Shotgun Approach

what does it do

what algorithm is used and what does it do

another method to see how clones overlap

find the physical alignment of clones

algorithm: minimal tiling path

allows you to tell where seqs are relative to each other