Techniques Use in GE

7/28/2019 Techniques Use in GE

1/41

TECHNIQUES USE INGENETIC ENGINEERING1


2/41

2

DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING


3/41

Analysis of DNA

gel electrophoresis- separates DNAfragments based on size

nucleic acid hybridization & probes

probes base pair with complementarysequences; used to detect specificsequences

DNA Sequencing reading the sequenceof nucleotides in a stretch of DNA

Polymerase Chain Reaction way toamplify DNA

3


4/41

DNA EXTRACTION

4

There are three basic and two optional steps in a

DNA extraction:

Breaking the cells open, commonly referred to ascell disruption orcell lysis, to expose the DNA

within. This is commonly achieved by chemical

and physical methods-blending, grinding or

sonicating the sample.

1
http://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Cell_disruptionhttp://en.wikipedia.org/wiki/Cell_lysishttp://en.wikipedia.org/wiki/Sonicationhttp://en.wikipedia.org/wiki/Sonicationhttp://en.wikipedia.org/wiki/Cell_lysishttp://en.wikipedia.org/wiki/Cell_disruptionhttp://en.wikipedia.org/wiki/Cell_%28biology%29


5/41

DNA EXTRACTION

5

Removing membrane lipids by adding a detergent

orsurfactants.

Removingproteins by adding aprotease (optionalbut almost always done).

Removing RNA by adding an RNase (often done).

Precipitating the DNA with an alcohol

usually

ice-cold ethanol orisopropanol. Since DNA is

insoluble in these alcohols, it will aggregate

together, giving a pelletupon centrifugation. This

step also removes alcohol-soluble salt.

2

3

4

5
http://en.wikipedia.org/wiki/Detergenthttp://en.wikipedia.org/wiki/Surfactantshttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/RNasehttp://en.wikipedia.org/wiki/Ethanolhttp://en.wikipedia.org/wiki/Isopropanolhttp://en.wikipedia.org/wiki/Isopropanolhttp://en.wikipedia.org/wiki/Ethanolhttp://en.wikipedia.org/wiki/RNasehttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Surfactantshttp://en.wikipedia.org/wiki/Detergent


6/41

DNA EXTRACTION

6


7/41

7

DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING


8/41

Gel Electrophoresis

Electrophoresis - the migration of chargedmolecules in an electric field though a solution or

solid support

Various types defined by support used

1. Paper amino acids, small peptides

2. Polyacrylamide Proteins, small DNA/RNA

(


9/41

Separation of charged molecules inan electric field.

Nucleic acids have 1 chargedphosphate (- charge) per nucleotide.means constant chare to mass ratio.

Separation based (mostly) on length:longer molecules move slower.

Done in a gel matrix to stabilize:agarose or acrylamide.

average run: 100 Volts across a 10 cmgel, run for 2 hours.

Stain with ethidium bromide:intercalates between DNA bases andfluoresces orange.

Run alongside standards of knownsizes to get lengths

Gel Electrophoresis


10/41

10

Gel

electrophoresis


11/41

Gel Electrophoresis


12/41

Gel electrophoresis uses a cross-linked

polymers (agarose) that contain various

pores.

Pores allow molecular sieving, where

molecules e.g. DNA, can be separated based

upon there mobility through the gel.

Gel Electrophoresis


13/41

Mobility = Charge + Molecular Dimensions

Charge per nucleic acid is

constant

This means separation is based

upon length of the DNA molecules

and this is how we can separateand identify DNA molecules.

Gel Electrophoresis


14/41

Linear DNA has a linear relationship todistance migration.

If add molecular markers of known mass

can calculate mass of our fragment by

plotting a linear plot.

Gel Electrophoresis


15/41

Other factors determining mobility-

1. Polymer concentration e.g. Agarose

2. Conformation of DNA

3. Electrophoresis

Gel Electrophoresis


16/41

Detection

1. Dye e.g. ethidium bromide

2. Audioradiography 32P,

3. Blotting (see later)

Uses

1. Analytical- Can determine size of DNAfragment,

2. Preparative Can identify a specific fragmentbased on size

Gel Electrophoresis


17/41

17

DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING


18/41

The idea is that if DNA is made singlestranded (melted), it will pair up withanother DNA (or RNA) with thecomplementary sequence. If one of theDNA molecules is labeled, you can detect

the hybridization.

Basic applications: Southern blot: DNA digested by a restriction

enzyme then separated on an electrophoresis gel Northern blot: uses RNA on the gel instead of

DNA

in situhybridization: probing a tissue

colony hybridization: detection of clones

microarrays

hydridization


19/41

Applications The main use of this technique is to identity any changes in

DNA sequencing or genes expressed, e.g. comparing genesexpressed by a diseased cell to genes expressed by anhealthy cell.

Other uses include- Testing for hereditary disease,Evolutionary history of species, Screening e.g.food

supply

Applications to synthetic biology

- identification of various parts in natural organisms,

-?more?

hydridization


20/41

Hybridization Process

All the DNA must be single stranded (meltat high temp or with NaOH). Occurs in ahigh salt solution at say 60oC.Complementary DNAs find each other andstick. Need to wash off non-specific binding.

Stringency: how perfectly do the DNAstrands have to match in order to sticktogether? Less than perfect matches willoccur at lower stringency (e.g. betweenspecies). Increase stringency by increasingtemp and decreasing salt concentration.

Rate of hybridization depends on DNAconcentration and time (Cot), as well as GCcontent and DNA strand length.

Autoradiography. Put the labeled DNAnext to X-ray film; the radiation fogs thefilm.


21/41

Labeling Several methods. One is random

primers labeling: use 32P-labeled dNTPs

short random oligonucleotides asprimers (made synthetically)

single stranded DNA template(made by melting doublestranded DNA by boiling it)

DNA polymerase copies the DNAtemplate, making a new strandthat incorporates the label.

Can also label RNA (sometimes

called riboprobes), use non-radioactive labels (often a smallmolecule that labeled antibodiesbind to, or a fluorescent tag), useother labeling methods.

h d d


22/41

Using specific probes that are labelled specific

sequences of DNA can be identified. There are three main hybridization techniques

which vary in the sample blotted and the

probes used;

1. Northern Blot-Transfer of an RNA sampleseparated and identified using DNA or RNA

probes.

2. Southern Blot-Transfer of an DNA sample

separated and identified using DNA or RNA

probes.

3. Western Blot- Transfer of an Protein sample

separated and identified typically using an

antibody.

hydridization


23/41

Blotting Transfer of DNA, RNA or Proteins, typicall

from a electrophoresis gel to a membrane e.g.

nitrocellulose. This membrane can then be subject to

further techniques such as hybridization.

Hybridization Process where two complementary

single strands of nucleic acid (DNA or RNA) form a

double helix.

hydridization


24/41

Southern blot

hydridization

24


25/41


26/41

In situ hybridization

26


27/41

27

DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING


28/41

Polymerase Chain Reaction (PCR)

A method for amplifying specific DNAsequences.

Components required:

- Target sequence

- A pair of primers- dNTPs (ATGC)

- DNA polymerase


29/41

Five noteworthy features of PCR:

1) The sequence of the target need

not be known.

2) The target can be much largerthan the primers (>10 kb).

3) Primers do not have to perfectly

match flanking sequences.

4) Stringency can be controlled by

temperature and salt (MgCl2).

5) PCR is very sensitive.



30/41

One PCR cycle involves three steps:

- Strand separation (95C)

- Hybridization of primers (54C)

- DNA synthesis (72C)

After n cycles, the sequence is

amplified 2n-fold.



31/41

PCR

31


32/41

Based on DNA polymerase creating a second strand ofDNA. Needs template DNA and two primers that flank the region to

be amplified. Primers are short (generally 18-30 bases) DNAoligonucleotides complementary to the ends of the region beingamplified.

DNA polymerase adds new bases to the 3' ends of the primers to

create the new second strand. go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA

from this region A key element in PCR is a special form of DNA polymerase from

Thermusaquaticus, a bacterium that lives in nearly boilingwater in the Yellowstone National Park hot springs. Thisenzyme, Taq polymerase, can withstand the temperature cycleof PCR, which would kill DNA polymerase from E. coli.

advantages: rapid, sensitive, lots of useful variations, robust (works even with

partly degraded DNA)

disadvantages: Only short regions (up to 2 kbp) can be amplified. limited amount of product made



33/41

PCR Cycle PCR is based on a cycle of 3 stepsthat occur at differenttemperatures. Each cycle doublesthe number of DNA molecules: 25-35 cycles produces enough DNA to

see on an electrophoresis gel. Eachstep takes about 1 minute tocomplete.

1. Denaturation: make theDNA single stranded by heating to94oC

2. Annealing: hybridize the

primers to the single strands.Temperature varies with primer,around 50oC

3. Extension: build thesecond strands with DNApolymerase and dNTPs: 72oC.



34/41

Other PCR Images


35/41

DNA Amplification in PCR

original DNA: very long moleculeswith neither end well defined.Number stays constant in thePCR reaction: no new ones aremade.

initial PCR product made fromoriginal DNA: has one enddefined by the primer, but theother end is not well defined.Copy number grows linearly.

all other PCR products have 2

ends defined by the primers, sothey have a constant length andcan be easily detected byelectrophoresis. Copy numbergrows exponentially.


36/41

36

DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING


37/41

DNA Sequencing

DNA Sequencing Determining the order of nucleotides

in a DNA molecule

Key technique as it can give us information about a DNA

molecule, e.g. location and order of genes, restriction sites.

In addition, for recombinant DNA gives verification of

gene cloning experiments.

2 possible uses for project Identify sequence of new part,

- Checking recombinant DNA.


38/41

38

Sanger

technique


39/41

Deoxyribonucleotide acid

This is essentially the monomer of

DNA. Polymerization of nucleotides

occurs by condensation reaction of a5 phosphate to a 3 hydroxyl group

Dideoxyribonucleotide acid

There is no 3hydroxyl group to

allow polymerization.

DNA Sequencing


40/41

DNA Sequencing


41/41

THANK YOU41

Techniques Use in GE

Documents

Transcript of Techniques Use in GE