techniques use in genetic engineering 1. 2 dna extraction electroforesis hybriditation pcr...

TECHNIQUES USE IN GENETIC

ENGINEERING 1

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DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING

Analysis of DNA• gel electrophoresis- separates

DNA fragments based on size• nucleic acid hybridization &

probes – probes base pair with complementary sequences; used to detect specific sequences

• DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA

• Polymerase Chain Reaction – way to amplify DNA

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DNA EXTRACTION

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There are three basic and two optional steps in a DNA extraction:

Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample.

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DNA EXTRACTION

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Removing membrane lipids by adding a detergent or surfactants.

Removing proteins by adding a protease (optional but almost always done).

Removing RNA by adding an RNase (often done).

Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.

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DNA EXTRACTION

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DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING

Gel Electrophoresis

• Electrophoresis - the migration of charged molecules 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 (<500bp)

3. Agarose – DNA/RNA

• Good preparative and analytical method

• Separation of charged molecules in an electric field.

• Nucleic acids have 1 charged phosphate (- 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 cm gel, run for 2 hours.

• Stain with ethidium bromide: intercalates between DNA bases and fluoresces orange.

• Run alongside standards of known sizes to get lengths

Gel Electrophoresis

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Gel electrophore

sis

Gel Electrophoresis

• 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

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 separate and identify DNA molecules.

Gel Electrophoresis

• Linear DNA has a linear relationship to distance migration.

• If add molecular markers of known mass can calculate mass of our fragment by plotting a linear plot.

Gel Electrophoresis

• Other factors determining mobility-

1. Polymer concentration e.g. Agarose

2. Conformation of DNA3. Electrophoresis

Gel Electrophoresis

• Detection 1. Dye e.g. ethidium bromide2. Audioradiography 32P, 3. Blotting (see later)

• Uses1. Analytical- Can determine size of DNA

fragment, 2. Preparative – Can identify a specific

fragment based on size

Gel Electrophoresis

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DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING

• The idea is that if DNA is made single stranded (melted), it will pair up with another DNA (or RNA) with the complementary sequence. If one of the DNA 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 situ hybridization: probing a tissue• colony hybridization: detection of clones• microarrays

hydridization

Applications• The main use of this technique is to identity any

changes in DNA sequencing or genes expressed, e.g. comparing genes expressed by a diseased cell to genes expressed by an healthy 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

Hybridization Process• All the DNA must be single stranded

(melt at high temp or with NaOH). Occurs in a high salt solution at say 60oC. Complementary DNAs find each other and stick. Need to wash off non-specific binding.

• Stringency: how perfectly do the DNA strands have to match in order to stick together? Less than perfect matches will occur at lower stringency (e.g. between species). Increase stringency by increasing temp and decreasing salt concentration.

• Rate of hybridization depends on DNA concentration and time (Cot), as well as GC content and DNA strand length.

• Autoradiography. Put the labeled DNA next to X-ray film; the radiation fogs the film.

Labeling• Several methods. One is random primers labeling: • use 32P-labeled dNTPs• short random

oligonucleotides as primers (made synthetically)

• single stranded DNA template (made by melting double stranded DNA by boiling it)

• DNA polymerase copies the DNA template, making a new strand that incorporates the label.

• Can also label RNA (sometimes called riboprobes), use non-radioactive labels (often a small molecule that labeled antibodies bind to, or a fluorescent tag), use other labeling methods.

• 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 sample separated 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

• Blotting – Transfer of DNA, RNA or Proteins, typically 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

Southern blot hydridization

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In situ hybridization

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DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING

Polymerase Chain Reaction (PCR)

• A method for amplifying specific DNA sequences.

• Components required:

- Target sequence

- A pair of primers

- dNTPs (ATGC)

- DNA polymerase

• Five noteworthy features of PCR:

1) The sequence of the target need not be known.

2) The target can be much larger than 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.

Polymerase Chain Reaction (PCR)

• One PCR cycle involves three steps:

- Strand separation (95ºC)

- Hybridization of primers (54ºC)

- DNA synthesis (72ºC)

• After n cycles, the sequence is amplified 2n-fold.

Polymerase Chain Reaction (PCR)

PCR

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• Based on DNA polymerase creating a second strand of DNA. • Needs template DNA and two primers that flank the

region to be amplified. Primers are short (generally 18-30 bases) DNA oligonucleotides complementary to the ends of the region being amplified.

• 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 Thermus aquaticus, a bacterium that lives in nearly boiling water in the Yellowstone National Park hot springs. This enzyme, Taq polymerase, can withstand the temperature cycle of 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

Polymerase Chain Reaction (PCR)

PCR Cycle• PCR is based on a cycle of 3

steps that occur at different temperatures. Each cycle doubles the number of DNA molecules: 25-35 cycles produces enough DNA to see on an electrophoresis gel. Each step takes about 1 minute to complete.

• 1. Denaturation: make the DNA single stranded by heating to 94oC

• 2. Annealing: hybridize the primers to the single strands. Temperature varies with primer, around 50oC

• 3. Extension: build the second strands with DNA polymerase and dNTPs: 72oC.

Polymerase Chain Reaction (PCR)

Other PCR Images

DNA Amplification in PCR • original DNA: very long

molecules with neither end well defined. Number stays constant in the PCR reaction: no new ones are made.

• initial PCR product made from original DNA: has one end defined by the primer, but the other end is not well defined. Copy number grows linearly.

• all other PCR products have 2 ends defined by the primers, so they have a constant length and can be easily detected by electrophoresis. Copy number grows exponentially.

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DNA EXTRACTION

ELECTROFORESIS

HYBRIDITATION

PCR

SEQUENCING

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 use’s for project – Identify sequence of new part, - Checking recombinant DNA.

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Sanger technique

Deoxyribonucleotide acid

This is essentially the monomer of DNA. Polymerization of nucleotides occurs by condensation reaction of a 5’ phosphate to a 3’ hydroxyl group

Dideoxyribonucleotide acid

There is no 3’hydroxyl group to allow polymerization.

DNA Sequencing

DNA Sequencing

THANK YOU

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