dna sequencing. * sequencing means finding the order of nucleotides on a piece of dna. * nucleotide...

DNA Sequencing

*Sequencing means finding the order of nucleotides on a piece of DNA . * Nucleotide order determines amino acid order, and by extension, protein structure and function (proteomics) . *An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal

*Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment DNA . *Technology is also extended to environmental and agricultural.

Methods of sequencing

1-Sanger dideoxy (primer extension/chain-termination) method: most popular protocol for sequencing, very adaptable, scalable to large sequencing projects

2-Maxam-Gilbert chemical cleavage method: DNA is labelled and then chemically cleaved in a sequence-dependent manner. This method is not easily scaled and is rather tedious .

*there are two main methods of DNA sequencing:

*Modern sequencing equipment uses the principles of the Sanger technique.

History of Sequencing

”Sanger Sequencing” developed by Fred Sanger et al in the mid 1970’s

Uses dideoxynucleotides for ”chain termination”, generating fragments of different lengths ending in ddATP, ddGTP, ddCTP or ddTTP

The Sanger Technique Principle : -The Sanger Technique uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc) These are molecules that resemble normal nucleotides but lack the normal -OH group.

-Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops. -Normally, this would be where another phosphate is attached, but with no -OH group, a bond can not

form and replication stops.

Requirements for Sanger Method

• DNA to be sequenced must be in single strand form.• The region to be sequenced must be 3’ flanked by

known sequence.• Reagents needed are:

– A primer complementary to the known region to start and direct chain synthesis. (15-30 nucleotides in length)

– DNA polymerase.– 4 deoxynucleotide triphosphates (dNTPs).– 4 dideoxynucleotide triphosphates (ddNTPs)

( small proportion ) .

Dideoxynucleotides

dATP ddATP

The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s, which terminates a DNA chain because a phosphodiester bond cannot form at this 3’ location

N

NN

N

NH2

O

HH

HH

HH

OPO

O-

O

POP-O

O

O-

O

O-

N

NN

N

NH2

O

HOH

HH

HH

OPH

O-

O

POP-O

O

O-

O

O-

DNA polymerase catalyzed nucleophilic attack of the 3’-OH on a phospho-anhydride

** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable to form a phosphodiester bond by nucleophilic attack on the phosphate, and it will cause a termination in the DNA chain

Mechanism of DNA polymerization

: :

O

HO

HH

HH

PO

O-

O-

O

HO

HH

HH

PO O-

Base

Base

O

HOH

HH

HH

OBase

O

P-O O-

O-

5’

3’

O

HOH

HH

HH

OPO

O-

O

POP-O

O

O-

O

O-

Base

O

HO

HH

HH

PO

O-

O

O-

O

HO

HH

HH

PO O-

Base

Base

O

HO

HH

HH

OBase

PO O-

O

HOH

HH

HH

OBase

O

P-O

O-

5’

3’

OP-O

O-

O

OHP

O-

O

Sanger Method• Partial copies of DNA fragments made with DNA

polymerase• Collection of DNA fragments that terminate with

A,C,G or T using ddNTP• Separate by gel electrophoresis

• Read DNA sequence

*The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up. *This stops replication on that piece of DNA . *The result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide. *Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined .

Sanger dideoxy sequencing: basic method

5’3’

5’ 3’

T T TT

ddA

ddA

ddA

ddA

ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand

Sequencing of DNA by the Sanger method

CCGTAC3’ 5’5’ 3’primer

dNTP

ddATP

GGCA

ddTTP

GGCAT

ddCTP

GGC G

ddGTP

GGGGCATG

A T C G

5’

3’

the sequence is complementary to the original strand

*Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel. *Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser. *Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment .

Gel Separation• The reaction mixtures are separated on a denaturing

polyacrylamide gel.– Denaturing to prevent the DNA from folding up on

itself while it travels through.– Polyacrylamide to separate the strands which differ in

length by only one nucleotide.• Each band corresponds to a sequence of DNA which was

terminated by a particular ddNTP.• This ddNTP is identified by lane in the radioactive method

and by color in the fluorescent method.• The lowest band on the gel is the shortest. The shorter the

strand, the earlier in the synthetic reaction the ddNTP was incorporated.

• The lowest band on the gel is at the 5’ end of our synthesized strand and is complementary to the 3’ end of our unknown fragment.

Sequencing Visualization Methods

• Two forms of labeling:– Radioactive

• Primer labeled (32P or 33P)• dNTP labeled (35S or 32P)

– Nonradioactive ( Fluorescence )*Primer labeled

*ddNTP labeled - ddNTPs chemically synthesized to contain fluorescence .

- Each ddNTP fluoresces at a different wavelength allowing identification .

Gel Visualization

• Radioactive method which requires four gel lanes, one for each reaction vessel.– Readout is done by hand or with a densitometric

scanner.• Nonradioactive fluorescence sequencing requires

only one gel lane because each nucleotide has a distinct color.– The readout process is done by laser scanner and

recorded by computer.

Nonradioactive ddGTP ddATP ddTTP ddCTP

Radioactive

Sequ

ence

of u

nkno

wn

frag

men

t

vs.Gel Electrophoresis and Readout of Reaction Products:

Sequ

ence

of u

nkno

wn

frag

men

t

Shortest synthesized band = 5’ end of synthesized strand

Longest synthesized band = 3’ end of synthesized strand

Manual vs. Automatic Sequencing Comparison list Manual Automatic

DNA labeling Radioisotope Fluorescence dye

Signal detect Film PhotomultiplierComputer

Sample 4 lanes /1 sequencing sample

1 lane /1 sequencing sample

Read length (av-erage)

250 500 bp∼ 400 1000 bp∼

Sample Output

1 lane

An automated sequencer

The output

Radioactive Primer Labeled Sequencing

4. dNTP’s (dATP, dGTP, dCTP, and dTTP)

ddGTPddATP ddCTP ddTTP

6. One type of ddNTP per reaction

Remember each reaction has many molecules each one incorporating its respective ddNTP and stopping at a different length.

7. DNA polymerase

3. Complementary primer, 5’end-labeled with 32P or 33P

5’

2. with region of known sequence

3’

1. Unknown fragment

5’

Reaction 2Reaction 1 Reaction 3 Reaction 4

5. Four separate reactions

5’ 5’

5’3’3’3’ 5’ 5’

5’

8. ddNTP incorporation -stops chain synthesis

3’3’ 3’3’

Radioactive Deoxynucleotide Labeled Sequencing

ddGTPddATP ddCTP ddTTP

6. One type of ddNTP per reaction

7. DNA Polymerase2. with region of known sequence

3’

8. ddNTP incorporation - stops chain synthesis

3’ 3’ 3’ 3’

3. Complementary primer

5’

1. Unknown fragment

5’

Reaction 1 Reaction 2 Reaction 3 Reaction 4

5. Four separate reactions

3’ 3’ 3’

5’ 5’ 5’

5’ 5’ 5’

4. dNTP’s 35S labeled dATP or dCTP

Fluorescent Primer Labeled Sequencing

5. dNTP’s (dATP, dGTP, dCTP, and dTTP)

ddGTPddATP ddCTP ddTTP

6. One type of ddNTP per reaction

What’s the big advantage here?

7. DNA Polymerase

1. Unknown fragment

5’

4. Fluorescent labeled primer. Different fluorescent dye per reaction

5’ 5’ 5’ 5’

8. ddNTP incorporation - stops chain synthesis

3’ 3’ 3’ 3’

2. with region of known sequence

3’ 5’

Reaction 1 Reaction 2 Reaction 3 Reaction 4

3. Four separate reactions

5’ 5’3’ 3’ 3’

Fluorescent Dideoxynucleotide Labeled Sequencing

4. dNTP’s (dATP, dGTP, dCTP, and dTTP)

ddGTP

ddCTP

ddTTP

ddATP

5. Fluorescent labeled ddNTP’s. Each labeled with a different fluorescent dye

Now we run our products on gel

6. DNA Polymerase

3. Complementary primer

5’

5’

5’

5’

Don’t forget that this and the all the previous reaction vessels have millions of our unknown fragment. Why do you think we’re only showing 4 representatives?

One reaction vessel

1. Here we have one reaction vessel, with four copies of our Unknown fragment.

5’

5’

5’

5’

2. A region of known sequence

3’

3’

3’

3’

7. Again ddNTP incorporation stops chain synthesis

3’

3’

3’

3’

• Strategy I– Four different reaction mixtures are set up– Primers covalently bonded to fluorescing dye at the 5’-terminus– Reaction products are separated by gel electrophoresis inparallel lanes– Laser induced fluorescence (LIF) is detected• Strategy II– Primers in each reaction mixture are labeled with differentfluorescent dyes– Reaction mixtures are mixed at the end of the reaction andseparated in a single lane by gel electrophoresis– The terminal base of each fragment is identified by thefluorescence of the dye on the associated primer

Commercial Sanger

• Strategy III– A single vessel used– Each of the ddNTPs is covalently bonded to a different fluorescent dye– The products are separated in single lane– The terminal base is identified by the characteristic fluorescence of the dye attached to the terminator

Commercial Sanger

Chemical Degradation Method

• Maxam-Gilbert method– 5’ end marked with radioactive label.

– Chemical reaction randomly cleaves strands of DNA at specific locations: G, A (some G), T (some C), C.

– Results in strands of varying lengths.

– Strands separated out with electrophoresis.

– Gels read with radioautography.

• Labeled DNA is treated with a reagent that cleavesDNA at a particular type of nucleotide .• Hydrazine cleaves DNA before every C-nucleotideat 1.5 M NaCl .• Reaction must have low yield so as to obtain random distribution of different length due to cuts at all the sites .• Labeled fragments are separated by SDS-PAGE .

Chemical Cleavage/ Maxam-Gilbert Method)

• Aliquot 1 Cleavage at only G– DNA treated with Dimethyl sulfate (DMS)– Methylation of G residues at the N7 position– the glycoside bond of the methylated G residue is hydrolyzed andthe G residue is eliminated.– Piperidine is added which reacts with hydrolyzed sugar residue,cleavage of the backbone results• Aliquot 2 cleavage at G and A– Use acid instead of DMS– Position of A revealed• Aliquot 3: cleavage at C and T– Treat with hydrazine, then piperidine• Aliquot 4: cleavage at C only– Treat with hydrazine in the presence of 1.5 M NaCl– Position of T revealed

Reagents for cleaving DNA

• Labeled DNA to be sequenced– 32P-ACCTGACATCG• Cleavage products– 32P-ACCTGACAT– 32P-ACCTGA– 32P-AC– 32P-A

Example of cleavage before C

Analysis of sequencing products:

Polyacrylamide gel electrophoresis--good resolution of fragments differing by a single dNTP– Capillary gels: require only a tiny

amount of sample to be loaded, run much faster than slab gels, best for high throughput sequencing

Comparison

• Sanger Method– Enzymatic– Requires DNA

synthesis– Termination of chain

elongation

• Maxam Gilbert Method– Chemical– Requires DNA– Requires long stretches of

DNA– Breaks DNA at different

nucleotides