Chapter 21 Manipulating Genomes Flashcards
What is the genome and what does it consist of?
All of the genetic material that an organism contains- as chromosomes and organelle DNA
2% coding DNA: exons
98% non-coding DNA: introns
What is the structure of DNA in the non-coding regions?
There will be repetitive base sequences
The number of times a particular sequence will repeat varies person to person
These repeats are collectively known as satellite DNA
What are VNTRs and STRs?
Variable number tandem repeats (minisatellites) : 20-50 base pairs that are repeated 50-100s of times
Short tandem repeats (microsatellites) : 2-4 base pairs repeated 5-15 times
How can VNTRs/STRs be used for DNA profiling? How does this relate to loci?
The number of repeats for each individual differs. We can compare the number of repeats between individuals to compare relations
When we say loci of an allele, its not base 100, after non-coding region 4
So it makes sense that you can have different lengths but all genes are in the same loci
What is the process of producing a DNA profile?
1) Extract the DNA
2) Use restriction endonucleases to cut DNA at specific recognition sites, which enable the STRs and VNTRs to remain intact. Then use PCR to scale up.
3) Use electrophoresis to separate the STRs/VNTRs by length. Shorter strands move further. In alkaline buffer to allow charge/current to be carried. Use sudden blotting to transfer to Nylon
4) Hybridisation of DNA: = Add fluorescent or radioactive DNA probes to bind to the DNA
5) Use X-rays or UV light to interpret the results
How do you interpret results from DNA profiling?
Compare the bands of each individual, if they alight, they are likely the same individual
If paternity testing, look at which bands are from the mother, so the remainder but be from the father
What are restriction endonucleases? What are recognition sites?
Enzymes with active sites complimentary to a specific base sequence, to a recognition site
Specific sequence of DNA bases which is complementary to a restriction enzyme
What are DNA probes?
Radioactive/fluorescent fragments of nucleic acid, complementary to a specific tandem repeat
What is PCR? What are common uses? And what is needed to carry it out?
Polymerase chain reaction, used to scale up the amount of DNA
e.g risk of disease and COVID resting, forensics, and paternity testing, detection o oncogenes, mutations
Needs original DNA , excess of free nucleotides, thermal cycle, and Taq polymerase (heat-resistant) and DNA primers
What are DNA primers and TAQ polymerase?
Primers: 20-30 bases complementary to DNA. Enables an easy site for DNA polymerase to bind to
TAQ polymerase: from thermophiles, heat-resistant and so does not denature at 92 degrees when DNA does
What is the process of DNA amplification with PCR?
1) Put the DNA, nucleotides, primers, and polymerase into the thermal cycler. Heat to 95 degrees celcius. The kinetic energy of the DNA molecules increases, vibrate faster, as the hydrogen bonds break, causing the denaturing of DNA into two polynucleotide stands
2) Cool to 55, DNA primers find complementary base sequence and anneal
3) Warmed to 72. So closer to the optimum for TAQ. Free DNA nucleotides form complementary base pairs, formation of phosphodiester bonds between adjacent nucleotides catalysed by polymerase, joining at the primers, forming DNA strands
4) Cycle repeats, 2-4-8-16-32…. DNA strands
What are the advantages of PCR?
Very quick- billions of copies made in hours
Does not require living cells, only the base sequence
What is the set up for gel electrophoresis?
Agarose gel on a support, with distinct wells for the DNA
Anode and cathode- so a power supply
Covered in an alkaline buffer solution to allow current to be carried
How does gel electrophoresis works?
Agarose gel enables electricity to be conducted and matrix helps trap DNA resisting movement, with buffer solution
As DNA is negatively charged, it is repelled by the cathode, attracted to the positive anode
Larger DNA fragments move more slowly, so separated by size
Stop electrophoresis before reaching the top
Southern blotting to transfer to nylon
Can be applied to nucleic acids and proteins
How does southern blotting work?
Place nylon on the gel
Covering in drying paper
As fluid uptaken, DNA fragments drawn up in exact positions to nylon
What briefly is the timeline for DNA sequencing?
1868: Mendel’s peas
1950: Chargass A=T C=G
1972: Fiers, 1st gene sequences
1975: Sanger sequencing
1996: Pyrosequencing
Now NGS, high throughput
How does sanger sequencing carried out and how does it work?
4 different reactions, each contains a different terminator base which prevents further nucleotides being added
1) Place the DNA, primers, nucleotides and small amount of a type of terminator base e.g T into a thermal cycler. Heat to denature DNA, cool and then heat to enable addition of nucleotides
2) Random incorporation of terminator nucleotides, and this process is repeated, every possible length of polynucleotide will be synthesised.
3) Repeat with A, C, and G terminators
4) Then use electrophoresis to separate by length, with each well corresponding to the base. Start from the one furthest away
How does high throughput sequencing differ from Sanger?
Similar approach but uses fluorescent terminator nucleotides so can take place in one cycler
Uses capillary electrophoresis with a laser for point illumination to identify the terminator bases
What is NGS?
Next generation sequencing
Enables sequencing of multiple DNA fragments at the same time
Flow cells
Nanopores and pyrosequencing
What is bioinformatics?
Developing and using computer software that can store and analyse biological data e.g base sequences
What is computational biology?
Using computers to study biology e.g modelling and simulations
What was the human genome project?
Aimed to identify all genes humans possessed
2 groups: 1st divided genomes into smaller sections, overlaps to help with positioning
2nd group cut up, sequenced then recombined
Share all data
What are the applications of gene sequencing?
Comparing between individuals of the same species and different species
Natural classification and evolutionary relationships
Prediction of amino acids/proteins that an allele codes for (with computational)
Identify risk factors for disease, and causes
Personalised medicine e.g Herceptin
Gene therapy
How has gene sequencing helped establish evolutionary relationships?
Enabled barcoding of a certain cytochrome base sequence to compare if an individual is part of a new species
Compare the base sequence to other organisms, more similar suggests a more recent common ancestor
Which in turn helps with natural classification
How can gene sequencing help predict proteins? Why might this fail?
Stores base sequence in a computer, use idea of triplets and universal code to enable a computer to predict the amino acid sequence and thus folding into the protein
More complicated: spliceosomes and removal in introns, some deletion/ modification of pre mRNA, activation, modification at golgi…
How can gene sequencing be used to help combat disease?
Find source of infection e.g how a virus has changed, spread, what treatment is possible
Identify faulty alleles e.g Huntington’s
What is synthetic biology and how can gene sequencing be used to help with it?
The design and construction of new biological entities e.g enzymes, GMOs, gene therapy…
Genes need to be sequenced first then put in , uses larger stretches of DNA
How can bioinformatics help determine whether a new allele causes disease?
Base sequence of the new allele and old allele should be placed in the database
Rapid comparisons should be made by software
Use modelling to predict the amino acid sequence from the triplets and universal code, to predict shape of molecules
Which can be used to predict if its function is impeded causing disease
What is genetic engineering? What is a GMO?
Manipulation of a genome
A transgenic organism, meaning it carries a gene from another organism
What are the main steps in genetically engineering a bacteria?
1) DNA sequencing and isolation of the desired gene.
2) Choose restriction endonucleases with recognition sites before and after the allele, ensuring overhangs, sticky ends, are formed. Need 1 enzyme for before the allele and one for after
3) Use the same restriction endonucleases to cut the plasmid, producing the same sticky ends.
4) Insert the gene into the plasmid. Hydrogen bonds form between the sticky ends and alleles. DNA ligase catalyses the formation of phosphodiester bonds in the sugar-phosphate backbone of the allele and plasmid
5) Transformation. Inserting the vector into the bacteria
6) Provide conditions for growth
What are different options for transformation of a vector and how do they work?
Heat in Ca2+, increases membrane permeability so plasmid can move in
Electroporation: small current to make pores, small holes, in the plasma membrane so plasmid can enter (not too high to prevent permanent damage)
Electrofusion: current applied to fuse two cells , nuclear membranes fuse so a polyploid with DNA from both- plants
How can we use promoters to control transcription of a gene we want to synthesis? Use the Lac Operon as an example
Aim to put the gene in between a promoter we know how to control and the structural genes of it
e.g Place between lac operator and lactase producing gene
Inhibitor molecule bound at promoter, when lactose present, released, then transcription can occur
So all we need to do is culture the bacteria in high conc of lactose and remove glucose
What happens to the engineered DNA? Where does it go?
Can be left as the plasmid and transcribed separately
Can incorporate to one of the chromosomes at a random or controlled place
Can be entirely new genetic material
What must be considered when synthesising a quaternary protein using genetic engineering?
Multiple alleles needed as each allele will code for one of the polypeptides only
Two separate genetic engineering projects
Then must bring the polypeptides together so they can associate and form the quaternary protein
Why do we use bacteria to synthesis insulin and not plants?
Changing 1 cell changes the entire genome of the organism
Replicates very quickly, large supply
How does genetically engineering a plant differ? What steps are needed?
Same up until transformation
Then electrofusion, with cellulases to remove cell wall, fuse, polyploid cell
Or agrobacterium tumafacients, engineer Ti plasmid, carried directly into the plants cells, forms a callus and eventually a whole plant
Instead of cutting out a useful gene, what other option is available to synthesise it?
Use mRNA of desired genes and use the enzyme reverse transcriptase to synthesise a single stranded complementary DNA molecule
How can GMOs be used for research?
Act as living records of DNA, DNA libraries
Act as models to show how changing a gene can affect an organism
Modified to help us better understand metabolism, drug resistance and vaccines
Why may crops be genetically engineered?
Add in resistance to herbicides
Add insecticides- e.g Bt toxin in soya
Enriched with vitamins
What are the advantages of genetically modifying plants?
Decrease need for pesticides reducing costs for farmers and helps the environment
Increase yield as less pest impact
Herbicides to kill weeds not plants
Could increase shelf life
Could increase nutritional value
Could enable growth in a variety of conditions
What are the disadvantages of genetically modifying plants?
Non-pests may be killed and reduced, reducing biodiversity
Promote insecticide resistance in pests
Genes transfer to wild populations, cross-breeding, disruption of food chains, super weeds
Allergic reactions to some products
Patenting issues- may cost farmers more
What are the issues relating to genetic engineering patents and general legal concerns?
May mean less economically developed countries do not have funds to access GMOs
Crops must be bought each year, seeds cannot be harvested for the next so pay each year
Lack of long term research
Without labelling GMOs removing choice
What is pharming? What are some examples of it?
Genetic engineering to produce human medicines
e.g creating models that contract disease
e.g produces useful molecules for pharmaceutical purposes
in an organism as may need modifications by golgi
What are some issues relating to pharming?
Strict ethical objections to promotion of disease and likely death of animals
How do we know putting these genes in will not cause harm?
What is gene therapy?
The mechanism by which genetic diseases are treated or cured by masking the effect of a faulty allele but insertion of a healthy allele
What are the two types of gene therapy? How do they differ?
Germ Line- of egg/sperm/zygote, permanent, effects future generations as alleles passed on
Somatic line- of adult cells, short-lasting, affects only those cells,
What are the two main vectors used in gene therapy and how do they work? Pros and cons?
Liposomes- phospholipids spheres with the plasmid inside, endocytosis by cell- not very efficient but unlikely to cause immune response
Viruses- edited so contains healthy allele, bind to specific receptors, DNA inserted into nucleus- more efficient and targeted but could cause immune response
What are some ethical considerations to gene therapy?
Germ line- can affect multiple generations, removing their choice- is something goes wrong
Slippery slope to designer babies
Potential impact unknown, interactions between other alleles, mutation rates…
Permanent- removing choice
