genetic modification techniques. tools of biotechnology collect dna restriction enzymes –blunt end...

Genetic modification techniques

Tools of biotechnology

• Collect DNA• Restriction enzymes

– Blunt end– Sticky end

• Ligase enzymes• Cloning• DNA carriers

– Bacteria– Virus– yeast

• Other tools to transfer DNA– Electroporation– DNA gun

• Host cells

Cloning

• NOT making an entire organism in this case

• Means using living organisms to reproduce large numbers of pieces of DNA

• So you put your piece of DNA into a vector, and the vector is called a clone

• But still also means lots of bacterial cells all derived from one cell

DNA carriers

Bacteria, viruses, yeast and bullets

Bacterial vectors

• Use the plasmids from bacteria

• Plasmids are removed from bacteria, cut with restriction enzymes, new material inserted and the DNA ligase enzymes seal the cuts

• Put back into bacteria, where it can be reproduced each time the bacteria divides– Produces many copies of the gene of

interest– Produces the protein associated with

gene of interest eg insulin– Engineered bacteria may have new

function

Viral vectors• Virus is string of DNA (or RNA) in a

protein coat• You can splice new genes into the

DNA of a virus and then put it back inside the protein coat

• If the virus is a bacteriophage (infects bacteria), it will then put the new DNA into the bacteria

• Bacteria then replicates the viral DNA

• Viruses are used to infect animal cells (animal cells are quite hard to engineer so viruses are used a lot)– Especially retroviruses (HIV)

• Viruses often only infect certain cells so you can use a certain virus to infect a particular type of cell– Eg lung virus to put better genes into

lungs of cystic fibrosis sufferers

Microballistics

• You can make really small bullets out of gold

• Gold is great because it doesn’t react with anything and it has high density so little bullets have mass

• If you coat the little bullets with DNA and shoot cells you sometimes hit the nucleus and the DNA gets integrated into the genome

• Microballistics is the only way to do genetic modification on grass

Electroporation

• Electrocute a cell which damages the membranes, and they then can take up DNA from the solution they are in

Host cell preparation

• Host cells are usually bacteria– Can be plants– Rarely animals because of ethics

• Prokaryotes and eukaryotes use different enzymes for transcription and translation of proteins, so can’t use one to the other transfers

Some other chemical stuff

DNA synthesis

• Scientists can now make their own one sided strands of DNA (called oligonucleotides)

• If you make two complimentary strands they will join together

• Why do it?– To make gene probes

• You make your sequence and see if it will join with the bit you are testing

• Can find mutations– Compare your oligonucleotide with the

natural strand of DNA

Automatic protein sequencing

• To make a protein you use DNA as the pattern for the amino acid chain

• So scientists want to know the sequence

• But the sequences are huge so they break the DNA into bits

• A machine does the work

Polymerase Chain Reaction

• Way to copy a little bit of DNA many times in vitro

• Cloning takes weeks, PCR takes hours

• Samples can be smaller and really old

• Done with PCR polymerase which is an enzyme that copies a template strand

• How to do PCR1. Heat up the DNA to 95˚C and it will

unwind and separate into two chains2. Add primer that matches the end

sequences of the DNA and cool to 40˚C so they anneal

3. Add lots of the four bases and some DNA polymerase and heat to 72˚C

4. The polymerase slides down the strand of single DNA adding the bases to make the complimentary strand

5. Repeat as required

Why do PCR?

• Forensic testing by police

• Anthropologists and achaeologists doing testing on fossils

• Gene testing for inherited diseases

• Testing for HIV and other viruses

• Cancer cell testing – bowel cancer

• Body identification

Gel electrophoresis

• Uses restriction enzymes which cut DNA into different lengths

• Each DNA sample produces different quantities of different lengths

• DNA is VERY NEGATIVE because of the phosphates in the nucleotides

• Having cut up your DNA you put in wells at one end of an agarose gel block

How to do gel electrophoresis

• Take your DNA and multiply the sample up with pcr

• Cut the DNA with restriction enzymes, every different sample will give different size bits of DNA because restriction sites will vary

• Put cut up DNA in well of agarose gel block

• Put block into machine that runs current through the gel

• The gel is like a net to the DNA, so little bits can move through it easier than long bits of DNA

• There is a positive electrode at the far end of the agarose gel which attracts the negative DNA

• Little bits of DNA travel further through the gel, and make a stripe in the gel, big bits don’t go far, and their stripe is closer to the well.

• Results are taken as photos

Interpreting the PCR

• If you share genes you will share restriction enzyme sites! So if you do a PCR on two people who share a lot of genes their stripe pattern looks the same

• You have to stain the DNA – ethidium bromide is commonly used, it fluoresces in UV light

• Can remove the DNA out of the gel, and it still works

Southern blotting

• Used to find genes of interest in combination with– Gel electrophoresis– Hybridisation

• There also exist northern and western blots

How to do it!

1. Cut DNA with restriction enzymes2. Separate DNA with gel

electrophoresis3. Take DNA from gel to a solid

support like nylon or nitrocellulose sheet by southern blotting

1. Gel is soaked in base to denature it2. Gel put on paper towel with ends in

salt solution

3. Then put a membrane on top of the gel, and put paper towels on top.

4. The towels will draw the salt solution through the gel and up into the towels

5. The dna comes off the gel and gets stuck on the membrane

4. A oligonuleotide which is radioactive is stuck on the genes – it only sticks to the genes of interest

5. X-ray film is laid on the membrane. The radioactivity leaves marks on the film

Sources of DNA/genes for cloning

1. Take them from the organisms and grow up fragments in bacteria. You have lots of bits of genome in different bacteria – called a genomic library

2. Complementary DNA• DNA has ‘introns’ which don’t seem

to code for anything (junk DNA) and exons that code for protein

To get rid of the introns use reverse transcriptase (an enzyme from retroviruses)

Put the edited mRNA into something un-named. Probably a bacteria – it will use the mRNA to make a strand of DNA, and then the single strand can be doubled using PCR

Has no control sequences (promotors) only exons

RFLP anlaysis

• Restriction fragment length polymorphisms

• Refers to the pattern you get when you cut up lengths of DNA and separate on a gel

• To see the pattern you have to1. Cut DNA with restriction enzymes

2. Gel electrophoresis

3. Southern blot

4. Use radioactive probe to find the genes of interest

5. Autoradiograph (take a photo with x-ray film)

– Used to find genetic markers, and inherited in Mendalian way

– Used for mapping genome, finding genetic disorders and for forensics

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