The genetic engineers toolkit A brief overview of some of the techniques commonly used.

These tools can be used in different ways depending on the job you are doing Size: Type:

Restriction enzymes (endonucleases) Restriction endonucleases cut the DNA at specific points called recognition sites (where there is a specific sequence of bases.) These enzymes were first found in bacteria as a defense against invading viruses They leave blunt or sticky ends. There are over 400 restriction enzymes so genetic engineers can cut DNA almost any where they want. they can cut out specific genes using restriction enzymes.

Blunt end restriction endonucleases Cut at specific sequence of bases leaving a blunt end. They are less specific

Sticky ends leave an overhang and are therefore more specific

Ligation or joining DNA using DNA ligase If DNA in a vector and DNA fragment are cut with the same restriction enzyme then the base pairs will match up and anneal ( base pair matching) and they can be joined together using ligase In the picture a recombinant DNA plasmid is being created.

PCR (polymerase chain reaction) This can take a small piece of DNA and copy it so you have lots (amplification)and it only takes a few hours It is divides into 3 stages *Denaturing the DNA Heating it up to separate the strands * Annealing- attaching a primer to the strand. * Extension- copying the template strand. Taq polymerase (which does not denature when it is hot) then completes the double strand.

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A PCR machine 535 × KB JPG

Gel electrophoresis This is a way of separating different DNA fragments out in a mixture which suspect committed the crime?

How does it work? DNA sample is cut up into different lengths during restriction digestion DNA is put into wells (holes) in a agarose gel matrix An electrical current is put through the gel Since DNA is negatively charged it moves towards the positive electrode. The DNA may be coloured or made radioactive so it shows up and is easy to see

It is a bit like fighting your way through the jungle. Big bits of DNA find it hard to get through and so travel slowly. Little fragments move faster.

Microsatellites DNA profiling uses electrophoresis of short tandem repeats (STRs or microsatellites) as the whole genome is too big! eg. CA CA CA CA CA CA CA CA CA CA CA CA A lot of DNA consists of long stretches of repeated nucleotides. These vary between individuals and can be separated using gel electrophoresis. Dna profiling usually uses about 10 STR’s

Reverse transcriptase in Gene cloning Making lots of copies of a gene or ultimately its protein product. This process may be done before PCR because it allows for the removal of introns so the DNA is shorter. DNA is transcribed into primary RNA. The introns are then removed and the exons joined together to make mRNA.

The central dogma of DNA DNARNA Protein Reverse Transcriptase turns this on its head !!!! It makes DNA from RNA. The DNA it makes will be the gene sequence of base pairs without the introns and any extra bits.