8B - Genome Projects and Gene Technologies Flashcards
What is a genome?
An entire set of an organism’s DNA, including all of the genes.
How does sequencing of an entire genome work and why?
- DNA is cut up into small sections, which are then sequenced and assembled again
- This is because sequencing methods only work on small sections
What was the Human Genome Project?
A project to map the entire sequence of the human genome.
What is a proteome?
All the proteins produced by an organism.
How and why does sequencing the genome of simple organisms help work out their proteome?
They don’t have much non-coding DNA, so the proteome can easily be determined.
When is proteomics useful?
Medical research (e.g. when identifying the antigens on the surface of disease-causing bacteria for vaccine creation)
Why is it hard to work out the proteome from the genome of complex organisms?
- They contain large sections of non-coding DNA and regulatory genes
- These do not code for particular proteins, so it is difficult to translate the genome into a proteome
How many human proteins have been identified so far?
30,000
Give some ways in which sequencing technologies are advancing.
- Automation
- Cost efficiency
- Large scale
Give an example of a development in DNA sequencing.
Pyrosequencing -> Allows 400 million bass to be sequenced in a 10 hour period
What does recombinant DNA technology involve?
Transferring a fragment of DNA from one organism to another.
How does recombinant DNA technology work and why?
- Genetic code is universal and transcription and translation mechanisms are similar too
- So DNA transferred to a different organism can be used to produce the same proteins (even in a different species)
What are organisms that contain transferred DNA called?
Transgenic organisms
Before DNA is inserted into another organism, what must first be done?
DNA fragment must be obtained containing the target gene
What are the 3 ways of producing gene fragments?
1) Reverse transcriptase
2) Restriction endonucleases
3) Gene machine
Describe how reverse transcriptase can be used to make a DNA fragment containing the target gene.
- mRNA is isolated from cells.
- Free DNA nucleotides and reverse transcriptase are added.
- The mRNA molecules can be used as templates for reverse transcriptase to make complementary DNA (cDNA).
What is the advantage of using reverse transcriptase to make DNA fragments containing a target gene?
mRNA created by the target gene is often more abundant than the two copies of the gene itself, so it is easier to obtain
What enzyme can be used to make a complementary DNA strand from mRNA?
Reverse transcriptase
What is the name for DNA produced by reverse transcriptase acting on mRNA?
Complementary DNA (cDNA)
Describe how restriction endonucleases can be used to make a DNA fragment containing the target gene.
- Some genes have a palindromic sequence of nucleotides either side. These are called recognition sequences.
- Restriction endonuclease is chosen that has an active site complementary to the recognition sequences.
- The sample is incubated with the restriction endonuclease, which causes the enzymes to cut the DNA via a hydrolysis reaction.
- This leaves the DNA segment with sticky ends.
What is the name for the place where a restriction endonuclease binds?
Recognition sequence
What makes a recognition sequence characteristic?
Have a palindromic sequence of nucleotides (i.e. with antiparallel base pairs)
Are there multiple different restriction endonucleases?
Yes, each one cuts at a different base sequence.
What type of reaction is a restriction endonuclease cutting DNA?
Hydrolysis
What are sticky ends?
- Small tails of unpaired bases at each end of a DNA fragment
- Created by restriction enzymes.
What are sticky ends used for?
Annealing the DNA fragment to another piece of DNA with sticky ends of complementary sequences.
What is a gene machine?
A technology used to synthesise DNA fragments from scratch without the need for a pre-existing template.
What is the advantage of using the gene machine to produce DNA fragments?
- Pre-existing template is not required
* DNA sequence does not have to exist naturally
Describe how a gene machine can be used to produce a DNA fragment with a target gene.
- DNA fragment is taken from a database or designed
- First nucleotide in the sequence is fixed to a support (e.g. a bead)
- Nucleotides are added step by step in the correct order.
- This involves protecting groups that make sure nucleotides are joined at the right points (to avoid unwanted branching)
- Short sections of DNA called oligonucleotides (about 20 based long) are made.
- These are broken off from the uppity and protecting groups, then the oligonucleotides are joined together.
What are protecting groups?
Groups added to a gene machine to prevent nucleotides from joining at the wrong points (which prevents unwanted branching).
What are oligonucleotides?
- Short sections of DNA
- About 20 nucleotides long
- Joined to make a full DNA fragment in a gene machine
What is amplification?
Producing multiple copies of a section of DNA.
Why is DNA amplified?
To make sure there is enough DNA to work with.
What are the two types of DNA amplification?
- In vivo cloning
* In vitro cloning
What are the stages of in vivo cloning?
1) Insertion of DNA fragment into a vector
2) Vector inserts DNA into host cells
3) Identifying cloned cells
What is a vector?
Something that is used to transfer DNA into a cell.
What are some examples of vector that can be used in in vivo cloning?
- Plasmids
* Bacterioohages (viruses)
In in vivo cloning, describe how the DNA fragment is inserted into a vector.
1) DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment. So the sticky ends are complementary.
2) The vector DNA and DNA fragment are mixed together with DNA ligase, which joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This is called ligation.
3) The new combination is called recombinant DNA.
What enzyme is used when joining a DNA fragment with a vector?
DNA ligase
What is recombinant DNA?
DNA which has been combined from multiple sources.
In in vivo cloning, describe how the vector transfers the DNA fragment into host cells.
1) If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
2) If a bacteriophage vector is used, the bacteriophage will infect the host bacterium by injecting its DNA into it. The recombinant DNA then integrates into the bacterial DNA.
How is a host cell persuaded to take in a plasmid vector containing recombinant DNA?
- The host bacterial cells are placed in ice-cold calcium chloride solution -> This makes their cell walls more permeable
- Plasmids are added
- The mixture is heat-shocked -> This encourages the cells to take in the plasmids
What solution is used to make the cell walls of bacterial cells more permeable so that they take up plasmids more easily?
Calcium chloride
What is heat-shock? Include temperature and time.
Heating to 42°C for 1-2 minutes
How can bacteriophages act as vectors?
They insert their DNA into the host.
What is the term for host cells that take up vectors containing recombinant DNA?
Transformed