Tools and Techniques for DNA Manipulation

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Tools and techniques for DNA manipulation

Gene technology is the artificial manipulation of genes

Gene technology’s use different techniques:

TOOLS:

Restriction enzyme
Enzyme that are used to cut DNA at specific sequences
‘Like a pair of scissors’
One of the most important tools in genetic engineering
They have the ability to cut DNA molecules at precise sequences of 4 to 8 base pairs called recognition sites A lot of bacteria are used to get restriction enzymes
Bacteria use restriction enzymes as a defence mechanism
We use over 1000 restriction enzymes today that engineers are able to: * isolate DNA
* sequence DNA
* manipulate individual genes
The site at which the DNA is cut may result in overhanging ‘sticky ends’ or overhanging ‘blunt ends’. Sticky ends only join to complimentary base sequence A restriction enzyme cuts a double stranded molecule at its specific recognition site You need to use the same enzyme to have the correct base pairs It is possible to use restriction enzymes that cut leaving no overhang – a so-called ‘blunt end’ DNA is cut in such a way it is able to be joined to any other end fragment but tends to be non-specific because they are no sticky ends as recognition sites – we won’t know what protein is produced, it is hard to cut this way because you need exactly the gene sequence because if you don’t then you change the sequence and amino acids produced.

Ligase
Join DNA fragments
DNA fragments produced using restriction enzymes may be reassembled by a process called ligation. Pieces are joined together using an enzyme called DNA ligase LIGASE IS AN ENZYME
DNA of different origins produced in this way is called recombinant DNA, because it is DNA that has been recombined from different sources The combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering Plasmid is a circular piece of DNA from bacteria

Two pieces of DNA are cut using restriction enzymes. We put the DNA pieces in bacteria to copy it Adding the ligase anneals (puts it together) the DNA backbone together

DNA probe
A fragment of complimentary nucleic acid that is labelled with a marker DNA probe is a small fragment of nucleic acid
Made by cloning or artificially synthesised – then labelled with an enzyme, radioactive tag or fluorescent dye tag The probe will bind to a complimentary DNA sequence by base pairing, identifying the presence and location if the target DNA RADIOACTIVE TAG: shows up as a dark band when the gel is exposed to photographic film FLUORECENT DYE TAG: shows up as fluorescent bands when gel is exposed to ultraviolet light source PROBE IS A SHORT SEQUENCE OF KNOWN DNA

GENETIC TECHNIQUES

Polymerase chain reaction (PCR)
* process that makes lots of copies of fragments of DNA or entire length of DNA Gel electrophoreses
* process where DNA fragments are stored according to size Southern blot
* process where probes are used to mark specific sequences involved electrophoreses

DNA amplification:
Using the technique called polymerase chain reaction (shortened to PCR), researchers are able to create vast quantities of DNA identical to trace samples. This process is also known as DNA amplification

Once we have amplified DNA, we can use it in various ways:
* DNA sequencing
* DNA profiling/fingerprinting
* Gene cloning (making copies)

Examples where you might not have enough DNA to do accurate testing: Crime scene, fragments of DNA from long extinct or single virus particle The laboratory process called the polymerase chain reaction or PCR involves the following steps each cycle

POLYMERASE CHAIN REACTION:
1. Separate strands:
Separate the target DNA stands by heating at 98degrees for 5 mins

2. Add reaction mix:
Add primers (short RNA strands that provide a startling sequence for DNA replication), nucleotides (A, T, G and C) and DNA polymerase enzyme.

3. Incubate:
Cool to 60degrees and...
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