manipulating genomes Flashcards
what is a genome
all of the DNA, of an organism including the genes, that carry all the information for making different proteins.
what does the term genome technology mean
investigate - PCR, electrophoresis, gene, DNA sequencing
profiling
- Manipulative genetic emerging and gene therapy
when might PCR technology be useful?
in order to make DNA in a crime scene (forensics)
what are the names of the units, that make up DNA?
nucleuotides
what are the complementary base pairs
- A + T
- G + C
the backbones (of DNA) run in opposite directions. what is the term we use for this?
antiparallel
What is PCR (polymerase chain reaction)
a biomedical technology, that can be used to amplify a short length of DNA, into thousands of millions of copies.
what are the 4 stages of PCR (basic)
- stage 1 - add the ingredients
- stage 2 - heat to 95 degrees.
- stage 3 - cool to 68 degrees
- stage 4 - heat to 72 degrees
why is TAQ polymerase used in PCR?
- Thermus aquaticus is a bacterium, adapted to live in hot springs
- therefore, they do not denature at high temperatures (95 degrees)
- thus meaning, we can recycle the DNA polymerase
how is PCR different, to natural DNA replication
- only short sequences, up to 10,000 bp (base pairs) can be replicated, not entire chromosome
what is electrophoresis
separation of DNA fragments, depending on their size. Can also be used, to sperate proteins by size.
what are the 3 components of electrophoresis
- agarose gel plate
- electrodes
- alkaline buffer
what is the purpose of the agarose gel plate
the smaller fragments, are able to travel further through the agarose gel, due to having a lower resistance to flow
what is the purpose of electrodes
at either end of the electrophoresis tank, as DNA will move towards the anode, as it has an overall negative charge
what is the purpose of the alkaline buffer
- there to keep PH in a narrow range,
- contains ions, that allow conductions of charge.
what is a restriction enzyme
cuts up DNA, at specific recognition sites.
give 2 uses of electrophoresis
- find the mother or farther of a child
- forensics evidence from a crime scene.
explain why the DNA sample is first treated with restriction enzymes
to cut the DNA into fragments
why do smaller fragments travel faster through the gel
lighter, less resistance to flow
what is the function of the loading dye
heavy, in order to sink to the bottom of the well and coloured, to allow you see the DNA.
what are the possible ways, other than the use of Dye, in which DNA, can be visualised after electrophoresis
- x - rays
- UV - light
- photographic film
what is the function of the buffer solution (electrolyte)
keeps a narrow PH range, provides ions that allows the conduction of charge.
what is one of the main problems, when separating proteins by Electrophoresis (SDS)
- some proteins, have a mix of a positive and negative surface charge, or a positive charge, which means they are not attracted to the anode.
what is the solution to the problem of Electrophoresis
- heat protein in order to denature the protein, and expose the hydrophobic region.
- add a negatively charge surface via SDS (sodium dodecyl sulphate)
give examples of when one may use SDS
- sickle cell anaemia, detect haemoglobin S protein, instead normal.
- thalassemia, leukaemia and aplastic anaemia , all have higher quantities of foetal haemoglobin.
what is a DNA probe?
a short single stranded piece of DNA (around 50 - 80 nucleotides long) that can bind (anneal) to a section of DNA being investigated
how can DNA probes be labelled
- use a radioactive marker (32P) (use film, to detect it’s presence)
- use a fluorescent marker (emits colour upon exposure, to UV light.
describe how a DNA probe works
- if a complementary sequence of DNA, is present the probe, will anneal to it.
- the radioactive/ fluorescent markers, allows us to detect it’s presence.
what is meant by annealing?
where two complementary DNA strands, join through the formation of H bonds.
how does a DNA microarray work?
- when a number of DNA probes, are placed on a fixed surface.
- DNA, for testing, is applied and if complementary for it is present, then it will anneal and be fixed to the probe
define a restriction enzyme (endonuclease)
an enzyme, that is used to cut DNA, at a specific recognition site (extracted from bacterial cells)
what is a restriction site?
certain sites, where DNA is cut.
what is DNA sequencing
working out the order of bases in DNA
what are the 3 methods of DNA sequencing
- chain termination method (or sanger sequencing).
- high throughput sequencing.
- genome sequencing.
what is in the reaction mixture (chain termination/ Sanger sequencing)
nucleotides, Taq Polymerase, DNA sample primers, + modified free nucleotides.
describe the difference between a normal and modified nucleotide and explain the effect that these have.
modified nucleotide, has no OH group, on the third carbon by adding a modified nucleotide, the chain is terminated.
what is the primer
a short nucleic acid
what is DNA polymerase
an enzyme needed, for DNA synthesis
what is an Activated Nucleotide
- has 3 phosphate groups, on binding, with a complementary base, they will lose two phosphates, through a hydrolysis reaction and release light.
what is ATP sulfurylase
an enzyme, that converts phosphates
what is APS
adenosine 5’ phosphatesulfate (need for the enzyme ATP sulfurylase
what is Luciferase
converts luciferin to oxyluciferin (generating visible light)
what happens to luciferin
converted to oxyluciferin
what is oxyluciferin
made from luciferin
what is the role of apyrase
enzyme, that degrades, unincorporated nucleotides, so it can be rerun with different nucleotides
what is genetic engineering
the process, that obtains a specific gene from one organism, and plate that gene, into another organism using a vector.
why is genetic engineering, also known as recombinant DNA technology
- because, you are combing the sources of DNA.
why genetic engineer?
- increase yield, by introducing traits, like pest resistance.
- improve crops, to have health benefits, i.e. vitamin enrichment.
- higher yield in animals.
- organ transplant.
- scientific research, e.g. GM rice.
what is Pharming?
- production of human medicines
- make animals models, like knockout mice, (gene deleted, so develop cancer).
- make human proteins ( insert human gene and promoter sequence, into fertilised egg - (e.g. desired protein in milk, etc.
what are the 4 key steps of genetic engineering (procedure)
- isolate desired gene
- get gene into vector (forming recombinant DNA.
- get vector, into cell (transformation.
describe how genetic engineering would take place in insulin (example case study)
- 1.) obtain desired gene - reverse transcriptase (ss cDNA -> dS DNA.
- 2.) - get gene into vector - ligase -> recombinant plasmids
- 3.) - transformation (get vector into cell) - combined with E. coli, and heat shock, treatment, with Ca, Cl ions.
- 4.) - E. coli, produces insulin protein
what is meant by the term transgenic
- an organism, that contains a gene from another species.
what is epigenetics
the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself.
what is the role of reverse transcriptase?
An enzyme, that catalyses the production of cDNA (complementary DNA) using an RNA template
what are restriction enzymes?
Endonuclease enzymes that cleave DNA at specific recognition sites.
what is the role of DNA ligase.
An enzyme, that catalyses the formation of phosphodiester bonds, between the sugar and phosphate groups, of adjacent DNA nucleotides.
what is DNA profiling
the creation, of a unique genetic fingerprint, of an individual.
what are minisatellites (VNTRs)
have a core repat limit of 20 - 50 base pairs (bp)
what are microsatellites (STRs)
have a core repeat unit, of 2-4 base pairs. these are commonly used, to create profiles, if they are smaller, and show a higher degree of variation in a populations profile.
what markers do we use to determine whether recombinant plasmid uptake in bacteria has been successful?
- antibiotic resistance.
- Fluorescence
summarise resitance in transformed bacteria (non - recombinant plasmid)
can growth on both ampaceyn and tetracycline
how would you use replica plating to work out successful colonies
the successful colonies, are the ones with the human genes, that only grow on ampicillin.
how are bacteria genetically engineered to minimise the risk?
- replicating plating, requires us to use antibiotic resistant bacteria.
- however, they have gene knocked out, meaning that they cannot make a particular nutrient, so can only survive on agar jelly in lab, when nutrient has been provided.
what is germ line gene therapy
gene therapy, by inserting functional alleles into gametes or zygotes.
what is gene therapy?
inserting a functional allele of a particular gene into cells, that contain mutated and non - functioning alleles. The persons, can them produce functional proteins, alleviating symptoms of their disorder.
what is somatic cell gene therapy
gene therapy, by inserting functional alleles into body cells.
what are the positives of germline cell therapy
- it only has to be treated once/it is permanent.
- all cells in body have the replaced (functional) gene.
- it can be passed on to offspring
what are the negatives of germline therapy
- there are ethical objections because of possible side effects to future generations.
- the are concerns if they could distract the expression or regulation onto genes.
- could it lead to patients, choosing desirable characteristics for their babies.
what is recombinant DNA
the transfer of fragments of DNA from one organism, or species, to another.
