Gene technology

Genomics: The study of the whole set of genetic info in an orgnaism's DNA in terms of the DNA bases. Placed on public access databases. 

Why we study DNA:
-Forensic fingerprinting/profiling
-Genetic Engineering 
-Gene Therapy
-Comparative gene mapping

Sequencing the genome:
1. Map out the genomes to identify exactly where it came from, i.e. what chromosome it is from 
2. Samples are mechanically broken into segments of 100,000 base pairs (SHOTGUN approach)
3. Inserted into bacterial artificial chromosomes (BACs) and transferrred to e.coli where clones are produced (clone library)
4. To sequence the BAC section, the cells containing the BACs are cultured 
5. DNA is extracted and cut by restriction enzymes>gives overlapping segments>electrophoresis to separate them>automated computer program reassembles the BAC segment sequence by analysing the overlaps. 

Why is comparative gene mapping important:

• Finding proteins in many living organisms indicates their importance
• Comparing pathogenic and non-pathogenic bacteria's genes to see which genes are causing the disease
• To determine evolutionary relationships 
• Compare DNA of individuals to see if there is a mutation 

Electrophoresis: 

-Can be used to separate fragments that are one base pair length difference
-Uses agarose gel that is covered in a buffer solution, with two electrodes at either end 
-The longer DNA fragments take longer to get to the positive electrode bc they get stuck in agarose gel
1. DNA sample treated with restriction enzymes
2. Placed into a well on the negative electrode side of the agarose gel
3. The gel is put into buffer solution and the electric current is passed through solution 
4. Because DNA have negative phosphoryl groups, they are attracted to +ve electrode
5. Longer fragments will take longer, shorter fragments get there first in the fixed time
6. The distance travelled can be shown up by dye.

Southern Blotting:
Nylon sheet placed over gel>tissue paper pressed on top and left overnight>fragments transferred to nylon and can be analysed 

Can be seen with a radioactive marker put on before+photographic film over the sheet. 

Short strand of DNA that is complementary to the section of DNA investigated. Labelled by:

Radioactive marking- that can be shown up under photographic film
Fluorescent marking- emits colour under UV light 

The probe will ANNEAL to the DNA section that it is complementary to. 

They are useful for:
-identifying genes to be used in genetic engineering 
-identifying the presence of a gene for a particular disease
-To identify the same gene in different different species in comparative gene mapping

Used to amplify small sequences of DNA-used in forensic profiling because it provides enough genetic material for genetic profiling

Differs from natural DNA replication:
-Uses primers
-separates the strands by heating and cooling, not the DNA helicase enzyme
-can only copy small strands of DNA not whole chromosomes

Method:-
1. DNA is mixed with free nucleotides and DNA polymerase (taq polymerase)
2. Heated to 95degrees, breaks H+ bonds that joined the two strands
3. Primers added+cooled to 55degrees so that they can anneal at either end of sample
4. Temperature raised to 72degrees (optimum for taq polymerase), and extends the double stranded section by adding free nucleotides
5. New DNA double stranded molecule generated
6. Process is cyclical and generates new DNA exponentially

Taq Polymerase is thermophilic-works in high temps. (thermus aquaticus)

Reaction mixture contains primers, DNA polymerase and free nucleotides- some of these carry a fluorescent marker w a specific colour. Once these are attached, the DNA polymerase is thrown off, and the the strand cannot have any further nucleotides attached.

1. Primer anneals to 3' end of the single stranded DNA
2. DNA adds free nucleotides to anneal to the DNA
3. If modified nucelotide added, DNA polymerase thrown off and strand has no more nucleotides attached 
4. Many different lengths+molecules of DNA made 
5. As the strands run through the computer, the laser reads the colour sequence of the nucleotide markers-sequence of bases
6. Lengths separated by electrophoresis

Basic steps:

1. Obtain the gene desired
2. Copy of this gene placed in vector
3. Vector carries it to recipient cell
4. Recipient expresses the gene through protein synthesis 

HOW?
Obtaining the gene- DNA probe sources, and restriction enzyme cuts it out, mRNA of genes can be used as template to make a copy, Gene synthesised by automated polynucelotide sequencer

Placing gene into vector- Using the enzyme DNA ligase, sealed into virus genomes/yeast cell chromosomes/vectors have regulatory sequences of DNA so the gene can be transcribed 

Getting gene into cell- Liposomes (wrapped in lipid molecules), Electroporation (high voltage pulse disrupts membrane), Viral transfer (vector is virus) 

Restriction enzymes (restriction endonuclease) cut through the DNA at certain points. Their restriction sites where they cut through a specific sequence are about 10 bases long. They catalyse a hydrolysis reaction that breaks the sugar-phosphate backbone of the DNA double helix which gives a staggered cut or sticky end (short run of unpaired, exposed bases that can anneal to other complementary sticky ends). 

When separate fragments of DNA need to be stuck together, DNA ligase catalyses the condensation reaction that joins the sugar-phosphate backbone.

In order to join fragments they must've been cut by the same restriction enzyme as this makes them complementary and allows the bases to pair up and anneal. DNA ligase seals the backbone.

DNA formed this way is called recombinant DNA (combining DNA from different sources in a single organism).

WHY?
-To improve feature of recipient organism, e.g. plants with herbicide resistance gene, livestock with myostatic gene
-Engineering organisms that can synthesise useful products, e.g. inserting gene for insulin in bacteria, gene for useful chemicals in sheeps milk and beta carotene in rice for Vit A. 

Bacterial cells and plasmids often used: 
Plasmid- circular piece of DNA separate from bacterial chromosome
If plasmids cut with the same restriction enzyme as the isolated gene, then when mixed, they can anneal by using DNA ligase to form recombinant plasmid. 

TRANSFORMED>TRANSGENIC
-Large quantities of the recombinant plasmid mixed with bacterial cells, some of which will take up the plasmid, less than a quarter of 1% though!! 
-heat shock and calcium salts increase the efficiency
-those that take up the plasmid are TRANSFORMED.
-this transformation means they contain new DNA-bacteria TRANSGENIC

Conjugation- exchanging genetic material from one bacteria to another. Plasmids often carry the gene for antibiotic resistance, and so if these are exchanged>antibiotic resistance between populations, e.g. MRSA. 

1. Enzyme makes nick in plasmid of donor cell. Conjugaton tube forms between donor and recipient.
2. Plasmid DNA replication starts in donor-free DNA strand moves through tube>recipient
3. Replication starts in recipient DNA>donor
4. Cells move apart and plasmid in each forms circle 

Allows for more genetic variety but it means the survival of resistant genes. 

• Scientists found the mRNA for insulin gene
• Using reverse transcriptase, complementary strand made 
• DNA polymerase and free nucleotides means second strand is made and copy of original DNA formed- cDNA 
• Unpaired nucleotides at each end to form sticky ends complementary to the sticky ends of the plasmid 
• Plasmid mixed with cDNA genes and sealed w DNA ligase to form recombinant plasmids.
• Mixed w bacteria and bacteria forms colony on an agar plate.

BUT NOT ALL TAKE UP PLASMID-HOW TO IDENTIFY THEM-REPLICA PLATING!

Original plasmids chosen because they have genes resistant to tetracyline and ampicillin. These genes are GENETIC MARKERS. Plasmids have restriction enzyme that cuts through tetracycline resistant gene-doesnt work. But ampicillin one does. 

Original plasmids chosen because they have genes resistant to tetracycline and ampicillin. These genes are GENETIC MARKERS. Plasmids have restriction enzyme that cuts through tetracycline resistant gene-doesnt work. But ampicillin one does. 

Replica Plating...
1. Bacteria grown on agar to form colonies
2. Some transferred to agar w ampicillin on-only those with the plasmid will grow
3. Some transferred to agar w tetracycline- those that didnt take up the plasmid that took up insulin gene will grow 
4. Identify the colonies you want to grow and produce them on large scale to make lots of insulin. 

-To combat Vit A deficiency-balanced diet-not easy for people in LEDCs 
-Rice contains the gene for beta-carotene (can be converted to Vit A in the gut), but it is in the green parts of the plant and not the grain which we eat.
-Found that the insertion of two genes (phytoene synthase and Crt 1) made the beta carotene gene present in the endosperm
-But the person would have to eat LOADS 
-cross breeding with other rice varieties allowed for more beta carotene to be produced, and the second version had 20times more in the rice.

Opposed because:- 
-human food safety unkown
-if bred with wild types, could contaminate them
-less biodiversity

Somatic cell gene therapy:
-Adding genes-Adding a functioning copy of a gene into the relevant specialised cells means that the polypeptide is synthesised and the cells can function normally
-Killing cells-using genetic techniques to make cancerous cells express antigens that alerts the immune system to attack them

Germline cell gene therapy:
-Engineering sperm, egg or zygote cells so that a copy of the engineered gene is expressed in the embryo-could be passed to offspring

Differences:
ST-functioning allele introduced to target cells-have to locate these/GLT-allele introduced into germline cells-straightforward
ST-treatment is short-lived and has to be repeated/GLT-all the cells will have this functioning allele-so might offspring
ST-Difficulties getting the allele into the genome in a functioning state-immune system attacks/ GLT-no difficulties but highly unethical-we dont know if the faulty gene will even be expressed
ST-gene manipulations restricted to person/ GLT- could be passed to offspring 

Xenotransplantation:
Between animals of different species
Allotransplantation:
Between animals of the same species 

Shortage in donors for organs and so pigs used>main obstacle is rejection, but they found they could engineer pigs to lack the a-1,3 transferase enzyme which triggers the immune system response. By turning this off, rejection would stop+save lives

Other issues w using pigs:
-Organ size
-Their lifespan is 15yrs-would age prematurely 
-Body temp is 39degrees, 2+ than humans

Ethics:
-Animal welfare don't agree on killing pigs to harvest organs
-Some religions (Jewish and Muslim) prohibit eating pork
-Poss disease transfer between humans and animals!