DNA Isolation, PCR & Sequencing

Dr. dr. Mgs. Irsan Saleh, M.BiomedBagian Farmakologi FK UnsriPalembangE-mail: irsan_saleh_hasani@yahoo.com

DNA Isolation

Blood Samples

DNA extraction

PCR amplification of the gene of interest

Analysis

Chromosome 17

RFLP Sequencing

AnalysisAnalysis

Micropipettors come in a range of sizes. They have disposable tips that hold tiny amounts of required reagents.

Lysate Fractionation by Density Gradients

When a solution of cesium chloride is centrifuged at extremely high speed, the cesium ions tend to sink slightly, forming a density gradient along the tube. Any other substance in the tube will float at the point where its density matches that of the gradient.

--> yields VERY pure DNA, but it takes very long time to perform

PCR Agarose gel electrophoresis

The final product UV visualisation

3-4 hours

Cell Debris Removal by Phenol/Chloroform Extraction

Mix thoroughly with an equal volume of organic solvente.g. phenol, chloroform, or phenol:chloroform

Centrifuge Collect aqueous phase

Organic

Aqueous

Interphase

The aqueous phase contains water-soluble molecules, including nucleic acids. Proteins and lipids become trapped in the organic phase, and are thus separated away. Insoluble debris becomes trapped in the interphase between the two layers

DNA can be Precipitated with Alcohols

• Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove residual salts and other contaminants.

• Discard ethanol and allow pellet to dry.

After

Add alcohol to precipitate nucleic acids from the aqueous fraction

Supernatant

Pellet

70% EtOH

Dissolve pellet (H2O, TE, etc.)

Before

Centrifuge Wash Centrifuge

--> also yields pure DNA, is cheap, but it involves hazardous chemicals and residual phenol can inhibit PCR and other enzyme reactions

Polymerase Chain Reaction (PCR)

DNA Sequencing – Dideoxy Method (Sanger’s Method)

• The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom.

• A dideoxynucleotide (dideoxythymidine triphosphate — ddTTP — is the one shown here) can be added to the growing DNA strand but when it is added, chain elongation stops because there is no 3′ -OH for the next nucleotide to be attached to.

• For this reason, the dideoxy method is also called the chain termination method.

Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops.

Normally, this wouldbe where another

phosphateIs attached, but with no -OHgroup, a bond can not form

and replication stops

The Sanger method requires• Multiple copies of single stranded template

DNA• A suitable primer (a small piece of DNA

that can pair with the template DNA to act as a starting point for replication)

• DNA polymerase (an enzyme that copies DNA, adding new nucleotides to the 3’ end of the template

• A ‘pool’ of normal nucleotides• A small proportion of dideoxynucleotides

labeled in some way ( radioactively or with fluorescent dyes)

• 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.

Steps Of Sequencing• The DNA to be sequenced is prepared as a single strand. This

template DNA is supplied with a mixture of all four normal (deoxy) nucleotides in ample quantities and a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color ddATP, ddGTP, ddCTP, ddTTP and DNA polymerase I.

• Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide instead of the normal deoxynucleotide.

• If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

• At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand.

• Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.

Model of DNA Sequencing Reaction

Inside the sequencer

Capillary tubes

Sample tray goes here

Reagents

Automated DNA Sequencing

Terima Kasih