Manipulating genomes Flashcards
What is DNA sequencing?
The process of working out the sequence of nucleotides within DNA.
What are the ingredients for DNA sequencing?
DNA for sequencing.
Primer
DNA polymerase
Excess of normal nucleotides
Terminator bases labelled with radioactive/fluorescent tags.
What do the terminator bases do?
They attach where a normal nucleotide would and stop DNA synthesis when they are included.
What do you need in each test tube for Sanger sequencing?
4 test tubes.
In each you need ;
The DNA being sequenced
A mixture of normal nucleotides in excess
One type of terminator nucleotide (ATC or G)
A primer
DNA polymerase
How can you then sequence the DNA in Sanger sequencing after the test tubes have produced lots of strands of DNA?
Eventually you will have built up all different lengths of DNA across the 4 test tubes.
If we visualise (gel electrophoresis) the fragments and line them up in order of length you can read the sequence from the terminator bases.
What is high-throughput sequencing?
A variety of approaches used to develop fast, cheap methods to sequence genomes.
Example is pyrosequencing.
What is PCR?
Polymerase Chain Reaction.
It can be described as the in vitro method of DNA sequencing.
What is PCR used for?
It is used to produce large quantities of specific fragments of DNA and RNA from very small quantities (even just one molecule of DNA or RNA).
Using PCR scientists can produce billions of identical copies of the DNA or RNA samples within a few hours, these can then be used for analysis.
How much DNA does PCR produce each cycle?
It doubles the DNA.
What is needed for PCR?
The target DNA being amplified.
Primers in excess.
DNA polymerase (Taq polymerase).
Free nucleotides.
Buffer solution.
Thermocycler to vary temperatures for the different stages.
What is the function of primers (forward and reverse)?
They are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied.
They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands.
They also prevent the strands from joining up again.
Why is Taq polymerase used in PCR?
It is thermostable, doesn’t denature at high temperatures needed for the PCR to occur (95oC)
What are the 3 key stages of PCR?
Denaturation
Annealing
Extension/elongation
What happens at the denaturation stage of PCR?
Separates the DNA into 2 separate strands.
The hydrogen bonds between the 2 strands are broken.
(95oC)
What happens during the annealing stage of PCR?
Adding primers to the 2 separated DNA strands.
Temperature cooled to 55-60 oC to help the primers bind to the DNA.
The primers signal to a polymerase where to start synthesising new DNA.
What happens in the extension stage of PCR?
New DNA synthesised.
2 Taq polymerase molecule attached to the 2 primers on the 2 DNA strands and move along the strand.
As they move along they create new “complementary” DNA by adding nucelotides.
72oC
What does bioinformatics do?
It stores and organises large amounts of data.
Facilitates access to data on DNA and amino acid proteins and databases of metabolic pathways as well as fast retrieval and sharing of information as well as algorithms and stats tests.
What is the format of bioinformatics?
It is universal.
What does bioinformatics allow scientists to do in terms of disease and genetic disease? What does this then allow?
Identify the source of outbreaks and potentially vulnerable populations in outbreaks (can then be useful in developing vaccines).
Can be used after sequencing to identify alleles that cause genetic disease.
This then allows computer modelling of new protein structure from the base sequence.
What is the definition of computational biology?
The study of biology using computational techniques.
What is computational biology needed for?
Analysis of large amounts of data.
What does computational biology actually do?
It uses the data stored in bioinformatics to build theoretical models of biological systems which can be used to predict what will happen in different circumstances.
What does computational biology allow for?
Rapid processing of data.
Prediction of amino acid sequences.
Algorithms and stats tests.
EG analysing DNA sequencing to work out 3D structures of proteins and for understanding molecular pathways such as molecular pathways such as gene regulation.
How does computational biology and informatics link together?
Computational biology can use bioinformatics to make predictions about the structure and function of a synthetic protein using DNA sequences.
What does whole genome sequencing determine? What does that mean for eukaryotes?
The complete DNA sequence of an organism’s genome.
For eukaryotes the is the genetic material of chromosomes, mitochondria and chloroplasts.
Where are genomes stored?
Gene banks.
What molecules are usually used to study evolutionary relationships?
Ribosomal RNA
Cytochrome C
Nuclear DNA
Mitochondrial DNA
How is ribosomal RNA used to study evolutionary relationships?
It is integral to protein synthesis and so it changes slowly, making the base sequence useful for showing connections between species that diverged long ago.
How is cytochrome C used to study evolutionary relationships?
It is a highly conserved protein involved in cellular respiration, so slight changes changes in the amino acid sequence can help identify evolutionary links.
How is nuclear DNA used to study evolutionary relationships?
Species that are more closely related will have more similar DNA sequences.
How is mitochondrial DNA used to study evolutionary relationships?
It mutates faster than nuclear DNA, so differences in sequences show the origin of species and their subsequent migrations.
What does analysing the genomes of pathogens allow scientists to do?
Doctors to find the source of an infection, for example bird flu or MRSA in hospitals.
Doctors to identify antibiotic-resistant strains of bacteria, ensuring bacteria are only used when effective.
Scientists to track the progress of an outbreak of any potentially serious disease.
Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic marker for use in vaccine.
What is DNA barcoding?
Technique used to identify particular sections of the genome that are common to all species but vary between them, so comparisons can be made.
What is proteomics?
The study of amino acid sequencing of an organism’s entire protein complement.
What is the definition of synthetic biology?
The synthesis of new genes/organisms.
What is synthetic biology used for?
Genetically modified organisms to produce drugs/enzymes/vaccines/useful molecules.
Synthesis of whole new organisms.
How is artimsinin an example of synthetic biology?
It is an antimalarial drug.
Until recently it was extracted from a plant.
Using synthetic biology scientists have created all the genes responsible for producing the precursor to artimesin.
These genes have been inserted into yeast cells and the yeast can be used to produce the drug.
Why might some people be concerned about the increase of synthetic biology?
Unknown consequences,
Ethical concerns.
What is pharmacogenetics?
By testing people’s genomes it is now possible to detect whether or not a drug will work.
Doctors can prescribe a drug that will work rather than the one fits all system.
Personalised medicine.
Big advances for breast cancer patients.
Why are non-coding regions used in DNA profiling?
Coding regions are too similar in most people.
What is satellite DNA?
Short sequences of DNA that are repeated many times. They are found in introns (non-coding regions).
