8.3-4 Genome Project and Gene Technologies Flashcards
What is a genome map?
A complete map of all genetic material in an organism (called the genome)
What are bioinformatics?
The science of collecting and analysing complex biological data such as genetic code
What is whole shotgun sequencing?
Many copies of the genome cut up into small fragments to be sequenced
Fragments are assembled using computer algorithms to match overlapping sections
What are SNPs
Single nucleotide polymorphisms
They are single base variations in the genome that are associated with disease and other disorders
Over 1.4 million in the human genome
How can SNP be used in biology?
In medical screening SNPs allow quick identification of potential medical problems
Allows for early intervention for treatment
What information can we apply from genomes?
Identification of potential antigens in order to produce vaccines
Sequence of amino acids in polypeptides can be predicted - allowing for synthetic biology
Why are complex organisms more difficult to determine the genome and proteome?
We have many non-coding genes
Introns
How does proteome differ in cells?
Did to selective gene expression not all genes are switched on in all cells therefore proteins aren’t always produced
Why is it easier to sequence the genome of a bacteria?
The prokaryotes have just one circular piece of DNA
With no Introns
How can the genome of an extremophile be useful?
If they can withstand extreme/toxic environments
They have a potential to clean pollutants or manufacture biofuels
What is Encode?
Encyclopaedia of DNA elements
It is a catalogue of regions of the human genome that control the function if genes
What can the human genome project help with?
Screening for mutated sequences and carriers
Screening for disorders before symptoms appear
What are some ethical considerations of the human genome project?
People being discriminated against
Misuse and ownership of genetic material
What is recombinant DNA technology?
It involves the transfer of fragments of DNA from one organism, or species, to another
Made possible due to DNA being universal
What ways can fragments of DNA be produced?
Conversion of mRNA to complementary DNA (cDNA)
Using restriction enzymes
Using a ‘gene machine’
How is mRNA converted to complementary DNA (cDNA)?
Isolate mRNA from cytoplasm using an ultracentrifuge
Using reverse transcriptase (found in HIV) mRNA acts as a template strand for cDNA to be formed
Using DNA polymerase double stranded DNA is formed from the single cDNA strand
What is a key point about mRNA being converted to complementary DNA (cDNA)?
There are no introns produced in the gene
(We took the genetic information from an already spliced mRNA strand)
So the can be cloned into prokaryotic bacteria - also no Introns
How are fragments of DNA produced from restriction endonucleases?
Restriction endonucleases are enzymes extracted from bacteria
They cut the DNA at specific sequences (enzymes are complementary) - and they cut to keep the entire gene intact
Best cuts = sticky ends (staggered like stairs)
The sticky ends allow two ends that are complementary to form a covalent bond and ‘rejoin’ (if the same restriction endonuclease was used)
What is the gene machine?
The allow genes to be artificially manufactured
Amino acid sequence of the protein | mRNA codons | Responding DNA sequence
In the gene machine what does a bacterial plasmid act as?
A vector
It carries one gene to another
What are gene markers? Types?
An extra gene in the plasmid that are easily identifiable
Antibiotic resistance genes
Fluorescence gene markers
Enzyme markers
How do antibiotics resistance genes work as markers?
If you cut a plasmid with a restriction endonuclease to insert a gene
You could cut through an antibiotic resistance gene
= the gene is no longer functional and no longer resistant to that bacteria
So the plasmids that are killed by a bacteria are the recombinant plasmids we want
Overview of replica plates in identification using antibiotic resistance?
The plasmids taken up by bacterial cells won’t all be recombinant
Culture the bacteria on an agar plate with some antibiotic in order to isolate the bacteria with plasmids taken up (they are resistant to it)
A replica plate is made and another antibiotic is added which should kill the recombinant plasmids
We have to then go back to the previous plate for the colonies killed on the second plate
Information about fluorescent gene markers?
The gene originates in jellyfish
Default plasmid = fluorescent
Cut plasmid with gene = not fluorescent
Advantage - easy to identify
Information about enzyme markers?
Enzyme in tact = colour change
Enzyme cut in a recombinant plasmid = no colour change
How is a recombinant bacteria cultured after it is formed? Factors?
Fermenters
Supply of O2
Condenser to regulate heat
pH regulator
(Don’t want to denature enzymes)
How do we ‘amplify’ DNA fragments after they are formed?
In vivo
In vitro
After a recombinant plasmid is formed what happens?
The recombinant plasmid is reintroduced into a bacterium
Using electroporation
It facilitates the uptake of the plasmid by increasing permeability of bacterial membranes = more chance of success
Achieved via addition of calcium chloride and rapid temperature change from 0-40 degrees
What is in vitro cloning?
Uses the Polymerase chain reaction (PCR)
In a thermocycler DNA fragments are separated into single strands by heating to 95 degrees - breaking hydrogen bonds
It is cooled to 55 degrees where DNA primers bind to the ends of the single strands
It is reheated to 72 degrees where DNA polymerase joins the nucleotides together forming a double stranded fragment using phosphodiester bonds and complementary base pairings - repeat around 30 times
Why are DNA primers used in PCR (in vitro cloning)? How many are needed?
It gives the polymerase something to bind to in order to start replication there
2 per fragment are needed as there are two strands formed with different base sequences
Why is 72 degrees used in the 3rd stage of PCR (in vitro cloning)?
It is the optimum temperature for TAQ polymerase to function at
TAQ polymerase is from a bacteria that live in hot springs
Why is in vitro cloning often used?
When you have a very small amount of DNA at a crime scene and you need a lot more to analyse
What is in vivo cloning?
Isolated DNA fragments are placed into plasmids by:
The plasmid and gene is cut using the same restriction endonuclease to create complementary sticky ends so the DNA and vector are complementary to join together
The fragments and plasmids are incubated together and if the fragment is taken up it is sealed using DNA Ligase forming phosphodiester bonds
A recombinant DNA molecule is created
What are the advantages and disadvantages of in vivo cloning?
Very accurate
Can introduce genes into different organisms - expressed
No risk of contamination
Slow - relies on bacteria regeneration time
Monitoring of cell growth
Needs a large sample
Needs more purification
What are the advantages and disadvantages of in vitro cloning?
Fast
Works on small quantities
Simple purification
Inaccurate
Risk of contamination
What are ethical issues of using recombinant DNA technology?
GM crops could affect food chains and could lead to pesticide-resistant weeds
Unknown long term effects of altering an organisms genome
What are two techniques that are important in identifying and locating particular alleles of genes in screening?
DNA probes
DNA hybridisation
What are DNA probes?
Short pieces of DNA (fluorescently or radioactively labelled - using P32 on the phosphate group)
That binds to a specific base sequence of the ‘faulty DNA’
The X-ray film will then be exposed
What can DNA probes be used for?
To detect heritable conditions of health risks
What should we be aware of with genetic screening?
Genetic councelling
Why can we do genetic fingerprinting?
Due to Variable number tandem repeats (VNTRs) - short repeating sequences
It is the 95% of DNA that doesn’t code for anything
This is UNIQUE to each individual
How is genetic fingerprinting carried out generally?
Gel electrophoresis - separates the DNA fragments to their size using electric current
It has: Sample wells: for the DNA Electrodes Buffered/isotonic solution Agar gel
How is genetic fingerprinting carried out specifically?
DNA is extracted (maybe undergoes PCR) and restriction endonucleases cut the DNA into fragments
They are separated in gel electrophoresis
The fragments are transferred to a nylon membrane - known as Southern Blotting
DNA probes are added and excess DNA rinsed
The radioactive probes are placed on x-ray film and development reveals dark bands only where DNA is
How and why does DNA move in gel electrophoresis?
DNA moves from the negative electrode to the positive electrode
This is due to DNA being negatively charged (because of the phosphate group)
What is significant about the position of the fragments in genetic fingerprinting?
The larger fragments are at the top - not moved as far from the well
The smaller fragments are further away
Due to the heaviness of the fragments
What are the main uses of DNA fingerprinting?
Forensics
Medical Diagnosis
Plant and animal breeding
Paternity tests
