AQA A2 Biology unit 5 - Gene technology

1) Reverse transcriptase

mRNA is used as a template to make lots of DNA

reverse transcriptase makes DNA from an RNA template -> the DNA produced it called complementary DNA (cDNA)

- DNA is isolated

- mixed with free DNA nucleotides and reverse transcriptase

- reverse transcriptase forms cDNA

- double stranded DNA formed using DNA polymerase and free nucleotides

2) Restriction Endonucleases

some sections of DNA have palindromic sequences -> restriction enzymes recognise these patterns

- DNA sample incubated with reverse transcriptase -> cuts DNA at specific sites

- leaves sticky or blunt ends

3) Polymerase Chain Reaction (PCR)

make millions of copies in a few hours

- reaction mixture set up containing; DNA sample, free nucelotides, primers, DNA polymerase

- mixture heated to 95 degrees = break hydrogen bonds between strands of DNA

- mixture cooled to 55 degrees = primers bind to strand

- mixture heated to 72 degrees = DNA polymerase lines up free floating nucleotides along template strand

- complementary strand formed

In vivo cloning: (using vectors)

- DNA fragment isnserted into vector -> vector DNA cut open using same restiction enzyme used to obtain fragement, mixed together with DNA ligase forms recombinant DNA

- vector transfered into host cells -> vector containing recombinant DNA encouraged to take up vector, host cells that take up vector are said to be transformed

- identify transformed host cells -> vectors contain marker genes e.g. antibiotic resistance, host cells grown on an agar plate containing an antibiotic, only genes containing the marker gene will survive and grow

In vitro cloning: (PCR)

- reaction mixture set up containing; DNA sample, free nucelotides, primers, DNA polymerase

- mixture heated to 95 degrees = break hydrogen bonds between strands of DNA

- mixture cooled to 55 degrees = primers bind to strand

- mixture heated to 72 degrees = DNA polymerase lines up free floating nucleotides along template strand = complementary strand formed

Benefits:

agriculture -> produce high yielding crops, more nutrious -> help combat malnutrition and famine e.g. golden rice, developed to reduce vitamin A deficiency 

industry -> some processes use biological catalysts, these can be transformed to produce large quantities for less money e.g. Chymosin, involved in cheese making

medicine -> drugs and vaccines produced from transformed organisms so they are produced in large quantiies and quickly, reduces costs. e.g. Insulin

Concerns:

agriculture -> monoculture, vulnerable to disease, possibility of 'superweeds' (resistant to herbicedes) 

industry -> people don't have a choice whether they want to consume food thats genetically engineered or not

medicine -> companies that own techniques may limit its use, prevent people from saving others lives, designer babies

genomes contain repetitive non coding sequences of DNA, different for each person -> can be compared to other individuals to determine genetic relationship

used in forensic science and medical diagnosis

electrophoreis

sample of DNA obtained from individual -> PCR used to make multiple copies of repeated sequences

end up with DNA fragments where the length corresponds to the number of repeats at a specific region

flourecent marker added to DNA fragements so they can seen under UV light

- DNA mixture placed in the agrose gel (conducts electricity)

- electrical current passed through gel -> DNA fragments negatively charged so the move towards the positive elctrode

- small fragments move further as they travel faster

- 2 genetic fingerprints can be compared to see genetic relationship

Dna probes

used to locate genes or to see if a person contains a mutated gene

short strands of DNA - specific base sequence thats complementary to target gene

binds/anneals to target gene if present in a sample

DNA probe is labelled so it can be detected

Restriciton Mapping (most genes too long to sequence -> so cut into sections and then put back together)

uses restriction endonucleases to sequence parts of DNA

1) different restriction enzymes used to cut labelled DNA into fragments

2) DNA fragments separated using elctrophoresis

3) size determines relative cut locations

4) resrtiction map of original DNA made

Gene sequencing

used to determine order of bases using the chain termination method

1) reaction mixture set up containing; DNA template, DNA polymerase, primer, terminators, free nucleotides

2) tubes undergo PCR -> strands are different lengths because they terminate at different points

3) DNA fragments are separated using electrophoresis

4) complementary base sequence can be read from the bottom up.

human diseases are caused by gene mutations

e,g. Sickle cell anemia -> mutation of heamoglobin gene, alters shape of red blood cells (makes them concave), can block capalliries -> lead to organ failure

however people who have this mutation are protected from malaria -> advantageous in Africa where its common

DNA probes & genetic screening (used to look for genetic disorders)

1) Probe is labelled and used to look for a single gene in a sample of DNA

2) probe used as part of a DNA microarray -> screens multiple genes at the same time

- glass slide with microscopic spots containing different labelled probes

- sample of human DNA washed over -> any attach to probes are seen as flourescent when under UV light, means they contain that mutated gene

genetic counselling -> advise patients and relaives about risks of certain genetic disorders, explain what positive results mean and possible methods of treatment

- involves altering defective genes -> how depends on whethe its caused by a dominant or recessive allele

- new alleles are inserted using vectors e.g. plasmids

2 types of gene therapy:

1) somatic = altering alleles in body cells e.g. cystic fibrosis, therapy targets epithelial cells lining the lungs

2) germ line = altering alleles in sex cells, means every cell of the offsrping will be effected by the therapy so they won't suffer from the disease

advatanges:                                                                       Disadvantages:

- prolong lives of people suffering from diseases             - effects of treatment short lived

- better quality of life                                                          - need multiple treatments

- people can have offpsring without disorder                   - difficult to get allele into specific gene

- decrease number of sufferers                                        - trigger immune response against allele