6.3 Manipulating Genomes Flashcards
Define DNA sequencing.
A technique that allows genes to be isloated and read.
What was FRed Sanger’s approach to Dna sequencing?
He used a single strand of DNA as a template for 4 experiments in separate dishes. Each dish contained a solution with 4 bases- A, T, C and G- plus an enzyme, DNA polymerase.
What did Fred Sanger develop?
He developed a methord that ultimetly allowed scientists to sequence whole genomes.
What did Sanger add to each dish when sequencing DNA?
To each dish, a modified version of one of the DNA bases was added. The base was modified in a way that, once inporperated into the synthesised compelmentary strand, no more bases could be added. Each modified base was also labelled with a radioactive isotope.
In Sangers experiment to sequence DNA, what was the added modified DNA bases labelled with?
A radioactive isotope.
In Sangers experimentz to sequence DNA, what did the modified DNA base cause once added to one of the dishes?
Once incorperated into the synthesised complementary strand of DNA, no more bases can be added.
What is generated as Sangers experiment to sequence DNA progresses?
Thousands of DNA fragments of varying lengths were generated.
In Sangers experimentz to sequence DNA, what happens to the DNA fragments? Why is this done?
The DNA fragments were passed through a gel by electropheresis. Smaller fragments travel further, so the fragments beome sorted by length.
In Sangers experiment to sequence DNA, what would be the first base sequence of a DNA fragment be if the first one-base fragment had thymine at the end?
T
What is this a picture of?
Previous was T
Fred Sangers experiment to sequence DNA.
What is Fred Sangers methord for sequencing DNA like?
The methord is efficeint and safe.
What did Sanger do with his methord to sequence DNA?
He used to to sequence the genome of a phage virus ( a virus that infecfts bacteria) called Phi-X174, the first DNA based organism to have its genome sequenced.
How did Sanger find the gemone of Phi-X174?
He had to count the bases, one by one, from the bands in a piece of gel- a very time consuming and costly process.
When cloning DNA, how isn a gene isolated?
By using restriction enzymes, from a bacterium.
How is DNA cloned for DNA sequencing?
- The gene to be sequenced must be isolated, using restriction enzymes, brom a bacterium.
- The DNA was then instered into a bacterial plasmid (the vector) and then into a Eshchichia coil bacterium hosts that, when cultured, divides many times, enabling the plasmid with the DNA insert to be coppied.
- Each new bacterium contained a copy of the candidates gene. These lengths of DNA were isolated using plasmid preparation techniques and were then sequenced.
When cloning DNA, how are multiples of the same single standed gene made?
The DNA is inserted into a bacterial plasmid (the vestor) and then into a Escherichia coil bactrium host that, when cultured, it divides many times, enabling the plasmid inserted to be coppied many times.
When cloning DNA, how is the coppied DNA in each bacterium removed?
These lengths were isolated using plasmid preparation techniques and were then sequenced.
Based on Fred Sangers method, what was developed in 1986?
The first automated DNA seqencing machine.
What was different about the new automated DNA sequencing machine and Fred Sangers methord?
Flouresent dyes instead of radioactivity were based to label the terminal bases.
What is an autroadiogram?
In the automated DNA sequencing machine, The flouesent dyes glowed when scanned with a laser beam, and the light signiture was identified by the computer. This methord dispensed with the need for technicians to read autoradiograms.
What is pyrosequencing?
A methord developed in 1996 and uses sequencing by sythesis to carry out DNA sequencing.
By what prosess was Sangers method of DNA sequencing?
Chain determination.
What does the process of pyrosequencing involve?
It involves synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting, by light emission, which base was added at each step.
What is the 1st step of pyrosequencing?
- A long strand of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs with a nebuliser.
What is the second step of pyrosequencing?
The lengths are then degraded into single-stranded DNA. These are the template DNAs and they are immobalised.
What is the 3rd stage of pryosequencing?
A sequencing primer is added and then the DNA is incubated with enzymes and different substrates. Once one of the 4 possible activated nucleotides, ATP, TTA, CTP and GTP is added at any one time and any light that is generated is detected.
During pryosequencing, WHat enzymes and substrates is the ssDNA incubates with?
- DNA polymerase
- ATP sulfurylase
- Luciferase
- Apyrase
- Adenosin 5’ phosphosulfate (APS)
- Luciferin
What is happening is the 4th step, part a of pyrosequencing?
One activated nucleotide (nucleotide with 2 extra phosphoryl groups) such as TTP is, incorperated into a complementary strand of DNA using the strand to the sequenced as a template.
What is happening in the 4th step, part b of pyrosequencing?
As the activated nucleotide is incorperated into the complementray strand of DNA, the 2 extra phosphoryls are released as pyrophosphate (PPi).
What is happening in the 4th step, part c of pyrosequencing?
In the presence of APS, the enzyme ATP sulphurylase converts the pyrophosphate into ATP.
What is happening in step 4, part d in pyrosequencing?
In the presence of ATP, the enzyme luciferase converts to oxyluciniferase.
What is happening in step 4, part e of pyrosequencing?
Converting luciferase to oxylucinefase generates visable light which can be detected by a camera.
At the end of pyrosequencing, what is the light generated proportional to? What does this tell us?
The amount of light generated is proportional to the amount of ATP available at the end, indiciating how many of the same type of activated nucleotides were incorperated adjecently into the complementary DNA strand.
At the end of pyrosequencing, what happens to the unincorperated acivated nucleotides?
They are degraded by apyrase and the reaction starts again with a different nucleotide.
How fast is DNA sequenced in pyrosequencing?
1 million reads occur simultaneously, so a 20-hour run generates 400 million bases of sequencing information. Software packages assemble these sequences into longer sequences.
What is bioinformatics?
Science of collecting and analysing complex biological data such as genetic codes, e.g. DNA sequencing.
What has been specially designed to store and analyse bioinformatics?
Software packages.
What is the Human Genome Project?
An international project to chart the entire genetic material of a human being, completed in 2003.
How many genes do humans have, found in the Human Genome Project?
24,000
What does whole genome sequencing determine?
It determines the complete DNA sequence of an organisms genome.
In case of eukaryotic cells, what are stored in gene banks?
Sequenced genomes are stored in gene banks.
What is a edukarotes complete DNA sequence made up of?
The genetic material of the chromosomes, mitochondia and chloroplasts.
What became clear when the human genome was compared with those of other species?
A few human genes are unique to us.
We share 99% of our genes with chimpanzees, what does this tell us?
It shows that the genes that work well tend to be conserved by evolution.
What is an example which genes being conserved by evolution can be seen? How have we taken advangtage of this?
Insulin
Pigs and humans have similar genes for insulin, which is why, prior to genetically-modifying bacteria to make insulin, pig insulin was used to treat patients with diabetes.
What does it mean if a gene is co-opted?
Co-option occurs when** natural selection finds new uses for existing traits**, including genes, organs, and other body structures.
What has happened to the gene FOXP2 in humans which makes us different from other mammals?
Tiny changes in the gene (co-option), means that in humans this gene allows speech.
Many differences between organisms are not because the organisms have totally differennt genes, what could this be because of?
Its because some of their shared genes have been altered and now work in a subtly different way.
Some changes to** regulatory regions** od DNA that do not code directy for proteins have also altered the expression of genomes.
How can changes in the regulatory regions of DNA alter the expression of genomes?
Regulatory and coding genes interact in such ways that, without increasing the number of genes, the number of proteins made may be increased.
Comparing genomes of different organims is good to look at relationships, what can this lead to?
Organisms being reclassified.
How can an extinct animals evolutionary relationship be verified?
The DNA from the bones and teeth of some extinct animals can be amplified and sequenced.
How may humans be genetically different?
Humans all have the same genes, but we have different alleles. 0.1% of our DNA is not shared with others.
All humans are genetically similar, what is an example of a rare exeption for this?
In rare cases, where a gene is lost by deletion of part of a chromsome.
Why is it significant that we dont share about 0.1% of our DNA with others?
Although 0.1% sounds small, our genome contains 3 billion DNA base pairs, this means that 3 million places on the DNA length where our DNA sequences can differ, due to mutations, such as substitution.
What are the places on out DNA where substitutions occur called?
Cauing variation
Single nucleotide polymorphisms (SMPs).
what is single nucleotide polymorphosisms (SNPs)?
Places on DNA where substitutions occur.
What is the effect of SNPs?
Some have no effect on the protein, some can alter a protein or alter the way a piece of** RNA regulates** the expression of another gene.
What process plays a major role in regulating gene expression in eukaryotic cells?
Methylation
What can methods to map mythelation of whole human genomes help researchers do?
Can help them understand the development of certain diseases, e.g. certain types of cancer and why they may or may not develop in genetically similar individuals.
What is epigenetics?
the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself, e.g. study of mythelation
Describe determining the sequence of animno acfids within a protein without having the organims genome.
Its a laborius and time consuming process.
How can researchers find the sequence of amino acids in a protein if they know the organims genome?
If they know the organims genome and know which codes for a specific protein, by using knowldge of which base triplet code for which amno acids, they can determine the primary structure of proteins. The researcherd need to know which part of the gene codes for introns and exons.
What is synthetic biology?
An interdisciplinary science concerened with designing and building useful biological devises and systems.
What sciences does synthetic biology concern?
- Biotenology
- Evolutionary biology
- Molecular biology
- Systems biology
- Biophysics
What may bge the ultmiate goals for synthetic biology?
To build engineered biological systems that store and process information, provide food, maintain human health and enhance the envitoment.
How is synthetic biology helped by DNA sequencing?
The sequences of DNA found by analysing genomes provides potential building blocks for synthetic biologists to build devices.
What are some examples of the application of synthetic biology?
- Information storage
- Production of medicines
- Novel proteins
- Biosensors
- Nanotechnology
What is the aplication of information storage from synthtic biology?
Scientists can encode vast amounts of digital information onto a single strand of sythetic DNA.
What is the aplication of production of medicines in synthtic biology?
Escherichia coli and yeast have both been genetically engineered to produce the precoursor of a good anitmalarial drug, arteminsinin, previously only available by exracting if from ceratin parts of the Artemisia plants at particular times in the plants life cycle.
What is the aplication of novel proteins in synthtic biology?
Designed proteins have been produced, e.g. one that is similar to haemoglobin and binds to oxygen, byt not carbon monoxide.
What is the aplication of biosensors in synthtic biology?
Modified bioluminecsent bacteria, placed on a coating microchip, glow if air is polluted with petroleum pollutants
What is the aplication of nanotechnology in synthtic biology?
Material can be produced for nanotechnology- e.g. amyloid fibres for making biofilms- for functions such as adhesion.
What is bioethics with synthetic biology?
Synthetic biology raises issues of ethics and biosecurity.
Synthetic biology is not about making synthetic forms from scratch, but is a potential for new systems with rewards and associated risks to be managed.
Define electrophoresis?
Process used to separate protein or DNA fragments to different sizes.
