(6) Manipulating Genomes Flashcards
Uses of genetic fingerprinting
Finding criminals
Finding fathers
Proving pedigrees
Revealing remains
Establishing evolution.
DNA fingerprinting
Restriction enzymes re produced by bacteria, they cut DNA at specific nucleotides to produce DNA fragments.
DNA probes are short single stranded pieces of DNA which are complementary to specific sequences in the genome. DNA probes are used to locate DNA fragments. They can also locate specific genes which can confirm diagnosis of genetic disorders. Probes are labelled with fluorescent dyes. Binding of probe to strand is annealing.
Electrophoresis
DNA is extracted
DNA is cut into fragments using restriction enzymes, producing fragments of different lengths.
Fragments are separated by size using gel electrophoresis, the smaller they are the quicker they travel - they are negatively charged so travel towards the positive electrode.
DNA fragments are blotted from gel onto nylon membrane to make DNA fragments single stranded.
Hybridisation - nylon membrane is incubated with labelled DNA probes, anneal to any DNA fragments containing complementary sequence.
After washing off excess DNA a probe, the by,on membrane is exposed to X-ray film.
When the film is developed, fragments which have annealed to the probe show up as dark bands.
PCR
Polymerase chain reaction.
Used to amplify DNA.
Heat DNA to denature it into single strands - breaks hydrogen bonds holding the bases of the DNA strands.
Cool and add primers which anneal to complementary base sequences.
Heat to allow DNA polymerase to attach to the primers - attaches complementary free DNA nucleotides. Also copies each strand.
2 copies of the original strand are made.
Repeat the cycle u til enough DNA is made so a sample can be analysed.
DNA sequencing
Automated process which is based on interrupted PCR and electrophoresis.
DNA polymerase adds free nucleotides to the growing copy strand. If a modified nucleotide is added, the reaction stops at that template strand. This produced thousands of different sized copies of the original DNA fragment Wahid is tagged with a modified nucleotide of a specific colour.
Newly synthesised fragments are separated with electrophoresis.
They pass through a laser beam and the colour is detected by a photocell which feeds information into a computer.
Then prints out the signal peaks for each fragment.
Each peak corresponds to a DNA fragment, the sequence of peaks reveals the sequence of the original DNA sample.
Sequencing genomes
Helps scientists to understand the genetic basis of disease and genetic disorders.
Uses a plastic slide - flow cell, allowing millions of fragments to be sequenced at once.
Bioinformatics - development of software to organise and analyse the data that is being generated.
Computational biology - build theoretical models of biological systems to predict what will happen in different circumstances.
Genome is sheared into fragments.
Each is placed into a separate bacterial artificial chromosomes.
Then placed into E. coli and culture to make multiple copies.
DNA extracted is cut into smaller fragments using restriction enzymes, then separated by electrophoresis.
DNA fragments are sequenced using machines and the sequences of overlapping fragments are compared to work out the final genome sequence.
Genetic engineering
Isolating a gene for a desired characteristic in one organism and placing it into another using a suitable vector.
Vectors - plasmids, contain genes for antibiotic resistance (so stop them from killing the bacterial cell). They act as genetic markers.
Bacteria cell can take up plasmid DNA directly through their cell walls (transformation) or by genetic exchange between bacteria, ells (conjugation).
MRSA contains many different plasmids with many different antibiotic resistance genes.
Conjugation - bacteria cell with plasmid and bacteria cell without plasmid join by conjugation tube. The plasmid replicate and the Copy of plasmid transfers from one cell to the other. Then the cells separate.
Bacteria cells takes up plasmid DNA directly through its cell wall, increasing genetic variation.
Creating genetically modified bacteria
Restriction enzymes cut the DNA strands at a particular site. This leaves sticky ends.
The human insulin gene is inserted into the plasmid to make recombinant DNA using DNA ligase, which anneals sticky ends.
Recombinant plasmid DNA taken up by bacterium - transformation (electroporation allows the membrane to take up the plasmid , calcium ions are then heat shocked to encourage the uptake of DNA).
Genetically modified bacteria is identified and cultured in a fermenter to make millions of copies of cells and millions of copies of the plasmid.
The human insulin is extracted and purified, then used to treat diabetes.
Cell does not always take up the plasmid DNA - may have transformed bacterial cell with no recombinant plasmid or bacterium cell without plasmid at all.
Widespread antibiotic resistance, transferring mutant genes to other pathogenic microorganisms, gm pathogens being used maliciously for biowarfare.
Engineering different organisms
Prokaryotes - easier than eukaryotes, making insulin, antibiotics and human growth hormone and enzymes for industry.
Eukaryotes- uses vector, plasmid that has a useful gene included such as pesticide production or higher yield. Plant cells are deliberately infected with the transformed bacteria.
Cut leaf and expose to bacteria carrying weed killer resistance gene and antibiotic resistance gene. Expose leaf to an antibiotic to kill new cells that lack the gene. Allow callus to sprout shoots and roots. Then transferred into soil where they can develop into fully differentiated adult plants.
Animals - harder to engineer DNA of animals because the animal cell membrane is more difficult to manipulate.
Pharming - using genetically engineered animals to produce human medicines.
Xenotransplants - transplant between species, however may introduce viruses to the human population and there is animal welfare issues.
Pharming
Clone a human hormone gene into a plasmid vector next to sheep promoter. This is functional only in mammary cells so the protein product is secreted into the milk.
Inject recombinant plasmid into a sheep oocyte, the plasmid DNA will intergrate into the chromosomal DNA, resulting in the addition of the hormone gene into the sheep’s genome.
Oocyte is fertilised and imp,anted into a female sheep which gives birth to a transgenic sheep.
Obtain milk from the transgenic sheep which contains the human hormone.
Purify the hormone form the milk.
Animal welfare issues
Some people think we should nit manipulate DNA.
Gene therapy
Functioning alleles of human genes are added to human genomes containing dysfunctional alleles. Liposomes (artificial vesicles) are used to introduce functioning alleles into cells. Only used to treat recessive genetic disorders because they do not replace dysfunctional genes.
Somatic gene therapy - functioning alleles are placed into adult differentiated cells. Cystic fibrosis. Treatment has to be repeated regularly because the differentiated cells do not divide and the functioning allele is not inherited.
Germline gene therapy - functioning alleles are placed into embryonic cells. All adult cells in the individual produced by the embryo will contain the functioning allele and will be inherited by the offspring.
This is illegal and unethical in humans because it could cause unpredictable results and the process could be abused to andante favourable characteristics leading to ‘designer babies’.
Evaluation of GM crops
Strengths - can increase yield, extended shelf life, nutritional value can be increased, can be used to produce human medicines.
Limitations - non pest insects can be damaged by toxins, may spread to wild populations, superweeds, reducing commercial value, allergies.
