8B Flashcards
Sequencing Genomes
Gene sequencing methods only work when the DNA is cut into smaller fragments and then put back together in order to give the sequence of the whole genome. E.g. Human Genome Project
Sequencing proteomes - Simple organisms, E.g. bacteria
Simple organisms don’t have a lot of non-coding DNA (introns) therefore they’re relatively easy to determine their proteome from their genome. this can be helpful in medical research and development, E.g. identifying the protein antigens on the surface of disease-causing bacteria/viruses, helping vaccine development
Sequencing Proteomes - Complex organisms, E.g. humans
Complex organisms contain large sections of non-coding DNA, as well as complex regulatory genes (determining when the gene is switched on/off) therefore it is very difficult to translate their genome into their proteome, as its hard to find bits of DNA which code for the proteins - work is being done for this though
Developing new sequencing methods
In the past, sequencing methods were labour-intensive, expensive and could only be done on a small scale. Now, the methods are often automated, cheaper and can be done on a large scale. With these new techniques, scientists can sequence a whole genome much faster.
Recombinant DNA Technology
Recombinant DNA Technology involves transferring a fragment of DNA from one organism to another - due to DNA being universal, transcription and translation are very similar, the transferred DNA can be used to produce a protein in the cells from the recipient, and they don’t have to be the same species.
Methods for making DNA Fragments - using Reverse Transcriptase
Cells which produce a protein, coded from a target gene, will contain many mRNA that are complementary to the gene. Due to mRNA being easier to obtain, they can be used as templates to make lots of DNA fragments. Reverse Transcriptase makes DNA from mRNA, producing complementary DNA (cDNA)
Methods for making DNA Fragments- using Restriction Endonuclease
Some sections of DNA have palindromic sequences of nucleotides. Restriction Endonuclease recognises specific palindromic sequences (Recognition sites) and cut/digest the DNA at these sites. Different Endonuclease’s have different specific recognition sequences because the shape of the recognition site is complementary to the enzymes active site
What are palindromic sequences?
antiparallel base pairs which can be read the same in the opposite direction
What are sticky ends?
If the recognition sequences are present at either end of the DNA fragment you want, Restriction Endonuclease cuts the DNA, by Hydrolysis. Sometimes the cut leaves sticky ends - unpaired bases at each end of the fragment, which can bind/anneal the DNA to another piece of DNA that has complementary sticky ends.
What are blunt ends?
Restriction Endonuclease cuts the DNA Fragment at opposite base pairs
Why are Sticky Ends important?
- DNA cut with the same Restriction Endonuclease will have complementary sticky ends and so they can join together
- DNA Ligase is used to join the two ends by joining the Sugar Phosphate Backbone
What does DNA Ligase do?
joins two ends of DNA Fragments together by doing the Sugar Phosphate Backbone
Methods for making DNA Fragments - Gene Machine
Technology which can synthesise DNA Fragments from scratch without a pre-existing DNA template.
The sequence required is designed, if it doesn’t already exist, and the first nucleotide is fixed to some sort of support - a bead. Nucleotides are added step by step in order, including adding protecting groups, which helps the nucleotides join at the right points, and prevents unwanted branching. DNA sections (Oligonucleotides) roughly 20 base pair long, are produced. The protecting groups are removed and the Oligonucleotides can be joined together to produce a longer DNA Fragment
Gene Cloning
Gene Cloning involves making identical copies of a particular gene. It can be done by two techniques: In vitro and In Vivo. Both require the isolated gene, which can be produced by:
- Conversion of mRNA to cDNA using Reverse Transcriptase
- Cutting DNA at specific palindrome recognition sequences using Restriction Endonuclease
In Vivo Cloning - what does it mean?
Where the gene copies are made within a living organisms. as the organism grows and divides, it replicates the DNA, creating multiple copies of the gene
What is a Vector?
A vector is something that’s used to transfer DNA into a cell. They can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (Viruses that infect bacteria)
In Vivo Cloning - Part 1, making Recombinant DNA
The Vector DNA is isolated, and then cut using the same restriction endonuclease that was used to isolate the DNA Fragment containing the target gene. This ensures both the Vector and DNA Fragment have complementary sticky ends. They are both mixed together is DNA Ligase to join the sticky ends together - called Ligation. The new combination of bases in the vector is called Recombinant DNA
In Vivo Cloning - Part 2, Transfer of Vectors into Host cell
The vector (Plasmid) and the host cell (Bacteria) are mixed together in an ice cold solution containing calcium ions. the solution is then heated to 42 degrees for 2 mins to increase the uptake of plasmids into bacterial cells. bacteria which have taken up the plasmid are said to be transformed.
In Vivo Cloning - Part 3, Identifying transformed cells
Not all the bacteria will contain the inserted gene as only few bacteria take up the plasmid, or some plasmids would have closed up without incorporating the gene. To identify the cells which have taken up the Plasmid, Gene markers are added to a vector on a separate gene at the same time the target gene was added. The host cells are grown on agar plates, creating colonies allowing the gene markers to be identified
What three types of gene markers are there?
- Gives resistance to a specific antibiotic - adding the antibiotic to the colonies, and if the colony survives, it has taken up the vector and can be grown
- produces a fluorescent protein, which can be seen under UV Light - if the colony glows, it can grow
- produces an enzyme whose action can be identified
Producing proteins
if the transformed host cell produce the protein coded for by the DNA fragment, the vector must contain the Promoter region and the terminator regions to allow the RNA Polymerase to bind to the DNA and start producing mRNA, as well as telling the RNA Polymerase where to stop
In Vitro Cloning - What does it mean?
Copies of DNA Fragments are made outside of a living organism using PCR (Polymerase Chain Reaction) PCR can be used to make millions of DNA Fragments in just a few hours, ideal for investigating crime scenes and there is only a small piece of DNA present
What are Primers?
Primers are short, single strands of nucleotide sequences which is complementary to one end of the DNA Fragment. they produce the starting sequence for DNA Polymerase, as well as preventing two strands of DNA rejoining.
What is a Thermocycler?
A computer controlled machine that varies temperature over a period of time
PCR - Polymerase Chain Reaction
- DNA Fragments, Primers, Nucleotides and DNA Polymerase are placed in a vessel in the Thermocycler at 95 degrees, to break the H bonds of the DNA strands into two separate strands.
- The mixture is cooled to 55 degrees, allowing the primers to anneal to their complementary bases on the DNA fragments.
- the temperature is then increased to 72 degrees, an optimum temperature for DNA polymerase to add the complementary bases along each of the DNA strands
- the process is then repeated, doubling the number od DNA Fragments each time - EXPONENTIAL INC. IN QUANTITY OF DNA - INC. AT AN EVER INC. RATE
what is DNA polymerase?
An enzyme which joins nucleotides together. it is obtained from bacteria which live in hot springs so they have a high optimum temperature and a fast rate of reaction. This means it is THERMOSTABLE - so they do not denature at high temperatures.
In Vitro vs In Vivo
- Vitro is extremely rapid compared to Vivo
- Vitro doesn’t require living cells, Vivo does
- Vitro is sensitive, small amounts of DNA needed, whereas Vivo needs larger amounts of DNA (Less useful for forensic work)
- Vitro requires pure sample as any contaminant will also be copied, whereas Vivo has almost no risk of contaminant as only genes cut with the restriction endonuclease, so complementary sticky ends are taken up
- Vitro is less accurate and any errors would be copied, whereas Vivo is very accurate as cells have mechanisms for correcting any errors made when copying
- Vitro cloned genes are in solution and cannot directly produce proteins, whereas Vivo produces transformed bacteria which can be used to produce large quantities of protein
