6.3 manipulating genomes Flashcards
what is bioinformatics?
Development of software needed to organise and analyse raw biological data.
What is computational biology?
Using bioinformatics data to build theoretical models of biological systems.
How can DNA sequencing be used?
- analysing the human genome
- analysing the genomes of pathogen
- DNA barcoding
- search for evolutionary relationships
what is synthetic biology?
The design and construction of artificial biological pathways, organisms, or devices, or the redesign of existing natural biological systems.
What are examples of techniques for synthetic biology?
- genetic engineering
- biological systems in industry
- Synthesis of new genes to replace faulty ones.
- Synthesis of entire new organism
What is dna sequencing?
A technique that allows genes to be isolated and read
Outline Sanger’s DNA sequencing method.
- In four separate Petri dishes, add a solution containing four bases, DNA polymerase, primer and DNA.
- Add a terminator nucleotide to each dish.
- A variety of partially completed DNA fragment strands will be produced.
- Gel electrophoresis sorts the fragments by length.
What is Sanger’s DNA sequencing?
Where modified nucleotides are used during DNA synthesis to halt the process, which results in different length fragments being made.
How is DNA cloned?
- Gene isolated using restriction enzymes.
- DNA inserted into vector (bacterial plasmid)
- A vector is placed in a bacterium, which divides so that the plasmid is copied.
- Dna lengths from the plasmid are isolated when sequenced.
What is pyrosequencing?
- synthesising the single strand of dna that is complementary to the strand being sequenced.
- light emission is detected each time.
Outline the process of pyrosequencing.
- DNA is cut into fragments.
- DNA lengths degraded into ssDNA and immobilised.
- DNA is incubated with sequencing primer, DNA polymerase, ATP sulphurylase, luciferase, apryase and the substrate’s adenosine 5’ phosphosulphate and luciferin.
- One active nucleotide is incorporated into the complementary DNA strand.
- Two extra phosphates are released as pyrophosphate.
- luciferase converts luciferin into oxyluciferin in presence of ATP
- light generated + detected
What is the DNA sequencing machine?
A machine that uses fluorescent dyes to label terminal bases so that they glow when scanned with laser beam. The light is then identified by computer.
How can gene sequencing be used?
- To determine the complete DNA sequence of an organism’s genome.
- The comparison between species to see evolutionary relationships.
- To predict amino acid sequences
What are some examples of the application of synthetic biology?
- information storage
- medicine production
- novel proteins
- biosensors
- nanotechnology
What are some examples of the application of dna profiling?
- Forensic science.
- Disease analysis.
- Maternity or paternity disputes.
What are some examples of the application of PCR?
- Tissue typing.
- Detection of oncogenes or mutations.
- Identification of viral infections.
- Forensic science
- research
- monitor the spread of Infectious Diseases.
What does PCR stand for?
Polymerase chain reaction.
What are VNTRs?
Variable number tandem repeats
- They occur more than 1000 locations on the human genome.
Outline the process of dna profiling.
- Obtain dna from the individual
- DNA digested by restriction enzymes, which cut the DNA into fragments.
- gel electrophoresis separates fragments.
- Banding patterns are produced and then compared
What are short tandem repeats?
Long stretches of DNA made up of repeating elements within the introns of an individual’s genome.
Will people have the same short tandem repeates
- Only identical twins will have identical STRs
- Close relatives may have similar STR’s.
How is DNA cut?
Using restriction endonucleases, which hydrolyze the phosphodiester bonds in the DNA backbone.
What is PCR?
The artificial replication of DNA.
How are the DNA strands separated in PCR?
Mixture is heated to 94 to 96 degrees, so that the hydrogen bonds break, leaving two single strands of DNA.
What is the 3 stages of PCR?
- Separation of DNA strands
- addition of primers
- synthesis of DNA.
Why is Taq polymerase used in PCR?
It is from a thermophilic bacteria so its optimum temperature is 72 degrees.
How are primers added during PCR
At 68 degrees, the primers bond to one end of each DNA strand.
How is DNA synthesised in PCR?
72 degrees is the optimum temperature for Taq DNA polymerase, it binds to the end of the double stranded DNA and catalyses the addition of DNA nucleotides to a single strands in the 5’ to 3’ direction.
What are the advantages of PCR?
It is very rapid and efficient.
Doesn’t require living cells only a base sequence?
When is PCR used?
If there is a small sample size, it is. for the amplification of the sample.
What is gel electrophoresis?
A method of separating dna fragments by size.
How does gel electrophoresis work?
- DNA is placed into the wells of a gel plate covered in a buffer solution
- Electrodes are placed at each end of a tank and an electric current is applied.
As DNA has a negative charge, the fragments move towards the anode. The smaller fragments will travel faster and a banding pattern forms.
How is the DNA in the gel made visible in electrophoresis?
They are made visible using a coloured, fluorescent or radioactive marker, or a DNA probe.
What is a DNA probe?
A short single-stranded length of DNA complementary to the section of DNA being investigated. It is labelled with a radioactive or fluorescent marker.
What are the uses of DNA probes?
- Locate a specific gene loci.
- Identify the same gene and different genomes.
- Identify the presence or absence of a specific allele.
What is genetic engineering?
The manipulation of the genome.
What are the stages of genetic engineering?
- Desired gene isolated.
- Recombinant DNA formed.
- Transformation of vector into recipient cell.
In genetic engineering how is the desired gene isolated?
- Restriction endonucleases cut the required gene from the dna of an organism, leaving sticky ends.
- Reverse transcriptase is used to produce a single strand of cDNA from MRNA.
In genetic engineering how is the recombinant gene formed?
- Isolated gene is inserted into vector, which combines with host DNA to make recombinant DNA.
- The same restriction enzyme is used to cut plasmid open, so the sticky ends are complementary.
- DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands of DNA.
In genetic engineering. how is the vector transformed into the recipient cell?
- Heat shock treatment.
- Electroporation.
- Electrofusion.
What enzymes are involved in genetic engineering?
- Restriction endonucleases
- DNA polymerase
- reverse transcriptase
- DNA ligase.
What is heat shock treatment?
A way of increasing the permeability of the bacterial membrane by increasing temperature and culturing cells in calcium rich solution.
What is electroporation?
Where high voltage poses make the cell membrane porous as it is disrupted.
What is electro fusion?
Where an electrical current is applied to 2 cells, which fuses them and their nuclear membranes together to form a polyploid cell.
What are reverse transcriptase enzymes?
Enzymes found in retroviruses. They reverse the transcription of mRNA to synthesise cDNA, which is single stranded.
How can a gene be obtained for genetic engineering?
- Transcription to produce pre-mRNA
- Reverse transcriptase to produce cDNA.
What is the function of ligase enzymes?
To join the DNA backbone of the fragments using phosphodiester bonds.
What are restriction enzymes?
Enzymes found in bacteria and archaea.
- they are used to cut up foreign viral DNA to prevent viruses making copies of themselves.
- uesd to cut/isolate DNA
What are transgenic bacteria?
Bacteria that contain the recombinant dna from another organism.
What is germline gene therapy?
Where a functional allele is inserted into the gamete or zygote
What is somatic cell gene therapy?
Where the functional allele is inserted into a body cell?
How can you identify transgenic bacteria?
- Antibiotic resistance markers.
- Fluorescent markers.
- Enzyme markers.
How can antibiotic resistant markers be used to identify transgenic bacteria?
- Identify gene in plasmid that is resistant to the antibiotics tetra and AMP
- Introduce insulin gene to plasmid with antibiotic resistance midway through the gene for tetra resistance.
- Some plasmids will take up the gene whilst others won’t.
- The plasmids with the insulin gene won’t be resistant to tetra, only AMP. So they can be placed on plates with AMP and tetra to see which ones survive.
How can fluorescent markers be used to identify transgenic bacteria?
- Cosmids contain gene for green fluorescent protein, GFP.
- Desired gene added into the gene for GFP.
- If bacterial colony is fluorescent, it doesn’t have the gene for insulin.
How can enzyme markers be used to identify transgenic bacteria?
- Desired gene inserted into gene for an enzyme such as lactase.
- Lactase enzyme will turn a colourless substrate blue. The bacteria that remain on the clear substrate have designed have desired gene.
Outline the process of genetic engineering in plants.
- cut leaf
- isolate gene and make recombinant DNA with a plasmid. Place plasmid in bacteria.
- Expose leaf to bacteria with a weed killer resistance gene and an antibiotic gene.
- Allow bacteria to deliver genes into leaf cell.
- Expose leaf to an antibiotic to kill cells that lack the new genes.
- Allow surviving cells to multiply and form a clump called a callus.
- Allow callus to Sprout shoots and roots.
- Transfer plant into soil so it can develop into a fully differentiated adult plant.
Why may pathogens be genetically modified?
For medical and epidemiological research.
Why may microorganisms be genetically modified?
To store a living record of DNA of another organism.
What is pharming?
Genetically modifying animals to produce pharmaceuticals.
What are the uses of pharming?
- create animal models for developing treatments to disease
- create human proteins such as insuln in milk
Why can cloning animals be bad?
It increases susceptibility of the population to disease.
Why are patents difficult for less economically developed countries?
They may be prevented from using them as people must pay for the seeds each year.
What is a perceived pros of pest resistance?
- Increased yields
- protects the environment as pesticides aren’t needed
- helps poor farmers.
What are the perceived cons of pest resistance?
- Pests may become resistant
- reduced biodiversity
- toxins may affect non pest insects and insect eating predators.
Why the perceived pros of disease resistance?
Higher yields as fewer are prone to disease
What are the perceived cons of disease resistance?
genes could be transferred to wild populations to create superweeds.
What other perceived pros of herbicide resistance?
Increased yields
What are the perceived cons of herbicide resistance?
Reduces biodiversity and there is a fear of super weeds.
What are the perceived pros of extending shelf life?
Less food waste.
What are the perceived pros of genetically engineering the growing conditions of a plant?
It allows crops to grow in a wider range of conditions, as they will be flood or drought resistant.
What are the perceived cons of genetically engineering the nutritional value of a plant?
People may be allergic to the different proteins made by genetically modified crops.
What are the perceived pros of genetically engineering the nutritional value of a plant?
Vitamins can be added, which can be used to reduce monourishment.
What are the perceived cons of extending shelf life?
Reduces the commercial value and demand for crops.
