21 - Manipulating Genomes Flashcards
What type of people have identical DNA?
Identical twins
What makes up an organism’s genome?
All the genetic material it contains (i.e. in eukaryotes mitochondrial DNA would be included)
What is an exon?
Region of DNA which is expressed and codes for a protein
What is an intron?
Sections of DNA which don’t code for amino acids. Introns are removed during protein synthesis.
What is satellite DNA?
Short DNA sequences which are repeated many times
What is minisatellite DNA?
A region where 20-50 base pairs is repeated 50 to several hundred times
What is another name for minisatellite regions?
Variable Number Tandem Repeats (VNTR)
In what 3 parts of DNA are satellite regions found?
Introns, centromeres, telomeres
What is microsatellite DNA?
A region of 2-4 bases repeated 5-15 times
Is satellite DNA coding or non-coding?
Non-coding
What is another name for microsatellite regions?
Short Tandem Repeats (STR)
Satellite regions always appear in the same position on what?
Chromosomes
What is DNA profiling?
Producing an image of the patterns in the DNA of an individual
What are the 5 main stages of DNA profiling?
- Extracting the DNA 2. Digesting the sample 3. Seperating the DNA fragments 4. Hybridisation 5. Seeing the evidence
Roughly what percentage of human DNA is introns?
98%
What are telomeres?
Regions of DNA at the end of chromosomes which do not code for making proteins, and which shorten each time replication occurs
What are centromeres?
The part of a chromosome that links sister chromatids during mitosis
Why does the number of STRs an individual has vary?
Lengths are inherited
What regions of DNA are examined during DNA profiling?
Satellite DNA
What technique is used to create a larger DNA sample for profiling?
PCR (Polymerase Chain Reaction)
What occurs during the extraction stage of DNA profiling?
DNA is extracted from a tissue sample
What are 2 samples which could be used to provide DNA for profiling?
- Cheek swab 2. Blood sample
What does the cutting in DNA profiling?
Special enzymes called restriction endonucleases
What is the site where a restriction endonuclease cuts a sample called?
Restriction site
How many cuts does each restriction endonuclease make?
2- one through each strand of the DNA helix
Why are a mixture of different restriction endonucleases used during DNA profiling?
To produce a mixture of different intact satellite regions
What happens in the second stage of DNA profiling?
Restriction endonucleases are used to cut up the sample
What technique is used to separate DNA fragments in DNA profiling?
Electrophoresis
What technique is used to transport the DNA from the electrophoresis gel to the nylon membrane?
Southern Blotting
What is a DNA probe?
A short, single-stranded section of DNA that has a label attached to make it easily identifiable, and which attaches to a complementary DNA sequence under particular pH or temperature conditions
What do DNA probes identify?
Microsatellite regions
What happens in the hybridisation stage of DNA profiling?
DNA probes attach to complementary DNA fragments, and excess probes are then washed off
What 2 types of label are generally added to DNA probes?
Radioactive and fluorescent tags
Are radioactive or fluorescent DNA tags more commonly used today?
Fluorescent
What does the final result of DNA profiling look like?
A pattern of bars which represents an individual’s DNA profile
How are radioactive DNA probes identified?
The membrane is x-rayed
How are fluorescent DNA probes identified?
The membrane is placed under a UV light so that they glow
What are the 3 steps of PCR?
- Separating the strands 2. Annealing primers 3. DNA synthesis
How does electrophoresis work?
The gel box has an electric field with a positive and negative end. DNA is negatively charged so it migrates to the positive end.
How is fragment size identified using electrophoresis?
It is easier for smaller molecules to move through the mesh of the box, so these travel further
What happens in the 1st stage of PCR?
Temperature increased to 90-95⁰C for 30 seconds to denature DNA
What happens in the 2nd stage of PCR?
Temperature decreased to 50-60⁰C and primers anneal to ends of DNA
What do primers do in PCR?
Allows DNA polymerase to add bases to them to build up complementary DNA strands identical to original sequence
What enzyme is used in PCR?
Taq polymerase
What happens in the 3rd stage of PCR?
Temperature increased to 72⁰C for at least 1 minute to allow taq polymerase to add bases to primers
What are 3 uses of DNA profiling?
- Crime scene investigation 2. Paternity testing 3. Identify individuals at risk of genetic diseases
What was the original method of DNA sequencing?
Sanger sequencing
How is the DNA for sequencing generated in Sanger sequencing?
By entering DNA into a bacterial plasmid
What was one refinement of the Sanger sequencing process?
Swapping radioactive labels for fluorescent ones
What are 4 disadvantages of Sanger sequencing?
- Very expensive 2. Slow 3. Can only be done 900 base pairs at a time 4. Essentially just ‘shotgunning’
What are 4 advantages of Next Gen sequencing?
- Much cheaper 2. Much quicker 3. Can sequence whole genome at once 4. Multiple genomes can be processed at once
What 2 things sped up the human genome project?
- Development of newer, more powerful computers 2. Automation of sequencing techniques
What is a terminator base?
A modified base which stops DNA synthesis
What are terminator bases given?
Fluorescent tags
What 4 things is DNA for sequencing mixed with in the 1st step of DNA sequencing?
- Terminator bases 2. Excess of normal bases 3. Primer 4. DNA polymerase
What are the 4 ingredients placed into in the second stage of DNA sequencing?
A thermal cycler
What happens at 96 degrees celcius in DNA sequencing?
The DNA strands separate into single strands
What happens at 50 degrees celcius in DNA sequencing?
Primers anneal to end of DNA strands
What happens at 60 degrees celcius in DNA sequencing?
DNA polymerase starts to build up new strands
What is the importance of terminator bases in DNA sequencing?
As they are incorporated at random, many different fragments of DNA of different lengths are created, so after many cycles every possible DNA fragment with the reaction terminated at every base will have been created
How are DNA fragments separated in DNA sequencing?
By length using capillary sequencing (basically gel electrophoresis in capillary tubes)
What are the fluorescent markers on terminator bases used for?
To identify the final base of each fragment
What does the order of terminator bases in capillary tubes show?
The order of the complimentary strand of DNA, which can in turn be used to identify the sequence of the template strand
Where are fragments sequenced in next-gen sequencing?
On a plastic slide
What two things about genome sequencing have fallen over the years?
- Time to sequence 2. Cost to sequence
What is bioinformatics?
The development of software and computational tools needed to organise and analyse raw biological data
What are 3 examples of things developed in bioinformatics?
- Statistical tests 2. Mathematical modelling 3. Algorithms
What is computational biology?
Using computers and the processed data provided by bioinformatics to build theoretical models of biological systems
What are 2 things computational biology gives us a better understanding of?
- 3D structures of molecules such as proteins 2. DNA sequences
What is genomics?
Using DNA sequencing methods and computational biology to study genomes
What 2 things can genomics reveal patterns in?
- Vulnerability to diseases 2. Inherited DNA
Why is genomics not the miracle identifier for what causes most genetic diseases?
Most are polygenic anyway
What are 4 things sequencing pathogen genomes enables?
- Doctors to find source of infections 2. Doctors to identify antibiotic resistant strains of bacteria 3. Scientists to track progression of diseases and monitor potential epidemics 4. Scientists to discover regions of pathogen DNA for drug targeting or markers for vaccines
What has DNA sequencing allowed in the field of phylogeny?
Evolutionary trees to be built with previously impossible accuracy
How has DNA sequencing aided phylogeny?
Basic mutation rate of different species’ DNA can be calculated, so we can see how long ago 2 species diverged from a common ancestor
What is the challenge for scientists wanting to use DNA sequencing to aid species identification?
To get stock sequences for each species
What charge does DNA have and why?
Negative, because of the phosphate group
What is proteomics?
The study and amino acid sequencing of an organism’s entire protein complement
What evidence is emerging about the link between genomes and proteins?
The amino acid sequence is not necessarily what would be predicted from the genome sequence alone due to some genes coding for multiple proteins
What is removed from pre-mRNA?
Introns and, sometimes, some of the exons as well
What are spliceosomes and what do they do?
Enzyme complexes which join exons which are to be translated
How can spliceosomes affect what proteins are coded for?
They can join exons together in multiple different ways, so the same pre-mRNA could produce multiple different proteins
How can proteins be modified after translation?
Cutting and combining of polypeptide chains and addition of phosphate or carbohydrate molecules by other proteins
What is synthetic biology?
Design and construction of new and biological parts; redesign and novel use of biological things for useful purposes
What are 4 techniques of synthetic biology?
- Synthesis of an entire new organism 2. Synthesis of new genes to replace faulty ones 3. Use of whole or partial biological systems in industrial contexts (i.e. immobilised enzymes) 4. Genetic engineering
What is genetic engineering?
The direct manipulation of genes for practical purposes.
Better understanding of what 2 fields has enabled successful genetic engineering?
Genomics and proteomics
What is an organism which carries a gene from another organism called?
Transgenic or a GMO
What do the basic principles of genetic engineering involve?
Isolating a gene for a desirable characteristic from one organism and placing it into another using a specific vector
What is the first stage of genetic modification?
Isolating the desired gene
What is the main technique used to isolate a gene for genetic modification?
By using restriction endonucleases to cut out the desired gene
Why is the same restriction enzyme used to cut both the gene from the target and a gap in the vector?
To create complimentary ends in the plasmid and desired gene
What is a sticky end?
A sequence of unpaired bases on a double-stranded DNA molecule that readily base pairs with a complementary strand. In genetic modification, can be found on the plasmid and inserted gene
What is a blunt end?
Where the restriction endonuclease leaves no bit of single-stranded DNA, so the end piece of DNA is double stranded
What is another technique to isolate a gene for genetic modification apart from using restriction endonucleases?
Getting a bit of mRNA for the desired gene and using reverse transcriptase to make the desired gene
What is an advantage of using the reverse transcriptase method of isolating a gene for genetic modification?
Makes it easier to identify desired gene
What is the DNA produced using reverse transcriptase called?
cDNA (complementary DNA)
What are the most commonly used vectors in genetic engineering?
Bacterial plasmids
What is used to fuse a plasmid and cDNA?
DNA Ligase
What is recombinant DNA?
DNA produced by combining DNA from different sources- for example, combining a bacterial plasmid with cDNA
What is transformation?
Moving the gene vector into the bacterium during genetic engineering
What is electroporation?
A method to introducing recombinant DNA into cells. Brief electrical pulse is applied, creating temporary holes in the plasma membrane and making it more permeable to vectors.
What is a method for checking for plasmid uptake in genetic engineering?
Adding a green fluorescent protein marker gene to the plasmid before uptake so you can use UV light to check uptake
What was a previous method of checking plasmid uptake before GFP and why isn’t it used any more?
Giving the plasmid an antibiotic resistance gene so any bacteria which don’t take it up can be killed off. Obviously no longer used due to concerns about antibiotic resistance.
What is electrofusion?
Using an electrical current to merge two cells and their respective genomes
What is a cell known as when it has multiple, merged genomes?
Polyploid cell
What types of cell does and doesn’t electrofusion work on currently?
Works well on prokaryotes and plant cells, not so much on animal cells
What is electrofusion important in the production of?
Monoclonal antibodies
Is it easier to carry out genetic engineering in prokaryotes or eukaryotes?
Currently prokaryotes
What are 6 examples of useful substances currently produced by genetically modified bacteria?
- Insulin 2. Clotting factors for haemophilia 3. Human growth hormone 4. Antibiotics 5. Pure vaccines 6. Enzymes used in industry
What are 2 methods of genetically modifying plants?
- Electrofusion 2. Using the bacterium Agrobacterium tumefaciens
How can plants be genetically modified using electrofusion? (4 steps)
- Removal of cell wall using cellulases 2. Electrofusion to form polyploid cell 3. Use of plant growth hormones to create new cell wall 4. Callus formation
What are the 5 steps of genetically modifying plants using A. tumefaciens?
- Cut leaf 2. Expose leaf to bacteria containing desired gene and antibiotic resistance gene, and allow bacteria to deliver genes into leaf cells 3. Use antibiotic to kill off remaining bacteria and cells without new genes. Wait for callus to form callus 4. Allow callus to sprout roots and shoots 5. Transfer new plant to soil
What are 3 advantages of crop GM?
- Quicker growth 2, More nutrients in crops 3. Disease and pesticide resistance can be achieved
What are DNA libraries?
Where fragments of DNA created during DNA sequencing are stored in the genomes of microorganisms
What can DNA libraries serve as sources of DNA fragments for?
Further genetic engineering applications or further study of their function
GM microorganisms are used in research of what 3 things?
- Novel medical treatments 2. Industrial processes 3. Development of gene technology
Why are GM pathogens not generally used for research?
Health and safety of researchers and the public, concern it could be used for biological warfare
Where are most GM crops found right now?
The USA, there is only 1 in Europe
What are 4 examples of crops which have already been genetically modified?
Rice, cotton, soy, corn
What are 4 things crops have been genetically modified to be or do?
- Resist drought and extreme heat 2. Contain Vitamin A (golden rice) 3. Produce insecticidal toxin 4. Resist herbicides
Is there any contemporary debate about the use of GM microorganisms?
Not really apart from the modification of pathogens for use in warfare
What are 2 wide-scale hopes for GM plants?
- Feed expanding human population 2. Overcome environmental issues
What is an economic concern about the use of GM crops?
People can be mugged off by corporations using patent laws, especially those in less developed countries who theoretically benefit most from genetic modification
What are 2 vectors for animal tissue currently under research?
- Modified viruses 2. Microinjections (tiny particles of DNA covered in gold)
What are 2 examples of GM animals?
- Swine-fever resistant pigs 2. Faster growing salmon
What is pharming?
Genetically modifying animals to produce medicines and other useful substances for humans
What are the two aspects of pharming?
Creating animal models (i.e. giving animals diseases genetically so they act as models for human treatment), creating human proteins
What is somatic cell gene therapy?
Gene therapy by inserting functional alleles into body cells to replace faulty ones (in a born person)
What are 4 positives of human gene therapy?
- Can prolong lives of those with genetic disorders 2. Can give genetic disease sufferers better quality of life 3. If germ line, sufferers can have baby without fear it gets condition or cancer 4. If germ line, could decrease number of people with disorder
What are 9 negatives of human gene therapy?
- Effects of somatic may be short-lived 2. May need multiple treatments in somatic 3. Might be difficult to get alleles into specific body cells 4. Inserted allele could be overexpressed and lead to overproduction of protein in question 5. Could cause immune response to vector 6. If allele inserted in wrong place could cause problems such as cancer 7. Technology might not just be used for medicine i.e. could be for cosmetic treatment 8. Could do more harm than good 9. May be too expensive and may rob funding from other, more proven technologies
What are 3 drawbacks of somatic cell therapy?
- Sufferers can still pass it on to their kids 2. Only temporary as somatic cells have limited life, and are eventually replaced by stem cells with healthy allele 3. Difficult to get vector into cell and get gene expressed
What is germ line cell gene therapy?
Inserting a healthy allele into a germ cell (usually an ovum) or an embryo immediately after fertilisation as part of IVF
What are 3 ethical objections to germ line cell gene therapy?
- Done to unborn individual, and thus without consent 2. Effects not yet fully known or studied 3. May eventually allow people to choose desirable or cosmetic characteristics in their kids
what is a genome?
the entire genetic material of an organism
in DNA profiling we are looking at an individuals differences in ___
genomes
what two things does the genome contain?
introns and exons
what are exons?
DNA that codes for proteins
what percentage of our bodies DNA makes us different?
0.1%
when using DNA profiling do you look at differences in extons or introns?
introns
even though some difference are in exons like eye colour, for DNA profiling you look at introns which are the non coding DNA
in introns there are short sequences of DNA repeated many time called?
satellite DNA
what is satellite DNA?
the short sequences of DNA repeated many times in introns
what does STRs stand for and what are they?
short tandem repeats
much shorter sequences of DNA and are repeated less
what can satellite DNA be spilt into?
VNTRs and STRs
what does VNTRs stand for and what are they?
variable number tandem repeats aka minisatellite
long sequences repeated many times
what technique is used to separate fragments of DNA?
gel electrophoresis
how can satellite DNA be used in DNA profiling to tell people apart?
- different people have a different number of repeats
- so they have different satellite patterns
identical twins have the same ___
genome
what is the satellite DNA like in identical twins?
the same because they have the same genome
what can DNA profiling be used for? (3)
- identify a criminal (forensics)
- determining paternity
- genetic diseases
what does PCR stand for?
polymerase chain reaction
how can satellite DNA/DNA profiling be used to identify familial relationships?
people who are related will have similar satellite DNA
e.g.: paternity tests as a father and child will have similarities in satellite DNA
what does PCR do and give an example of when it can be used?
it is artificial DNA replication and can make lots of chains of DNA
for example: at a crime scene if only a small amount of DNA is present PCR can be used to replicate the DNA so it can be more easily analysed
describe the stages of DNA profiling after finding a small pool of blood at a crime scene?
1) extract DNA from the blood
2) might only be a small amount of DNA so use PCR to replicate the DNA and amplifies the DNA sample
3) enzymes called restriction endonucleases cut the DNA strands at the restriction sites so we get fragments of DNA
4)’ use gel electrophoresis to analyse the DNA and compare it to suspects
5) once the blocks are produced use southern blotting which is adding a nylon sheet on top of the agar gel plate so show them better so you can see the similarities
6) hybridisation: add a dye (fluorescent) which shows the blocks better
7) then you can see the evidence and similarities
where do the restriction endonucleases cut the DNA?
at the introns but ensure to keep the satellite DNA intact
explain the steps of gel electrophoresis?
- separates the fragments
- on an agar gel plate and uses electricity
- wells on the agar gel and the DNA fragments are added to the wells at the negative electrode
- this is because DNA is negative so it will repel and move from the negative electrode to positive electrode
- immerse the gel plate in alkaline solution
use southern blotting by adding a nylon sheet over the gel plate which will absorb the DNA fragments that were separated - hybridisation: fluorescent dye so the bands of fragments of DNA show up better
why is DNA negatively charged?
the phosphate group
in gel electrophoresis, why is the agar gel plate immersed in alkaline solution?
splits the double stranded DNA into single strands so they can be analysed more easily
what is gel electrophoresis in simple terms?
the use of electricity to separate DNA into its fragments based on their size and charges
why do we analyse introns instead of exons in DNA profiling?
because everyone’s genomes are very similar so it is most likely that introns are similar and will not give unique profiles
but exons being the non coding DNA will
why is PCR important?
increase the amount of DNA for further analysis (eg: from a crime scene which might not be much)
state the three stages of PCR?
- denaturation
- annealing
- synthesis
what temp does denaturation occur at in PCR?
95 degrees
what temp does annealing occur at in PCR?
50-60 degrees
what temp does synthesis occur at in PCR?
72 degrees
what is the first stage of PCR and explain it?
denaturation - occurs at 95 degrees
- because it is so hot the molecules gain KE so the H bonds between the double helix break
- forming two single strands of DNA
what machine does PCR occur in and what does it do?
thermo cycler - controls and changes the temperature at specific times in the different stages of PCR
what is the second stage of PCR and explain it?
annealing - occurs at 55 degrees
- have primers which are short DNA sequences that bind the the start of the gene you want to amplify
- this is because DNA could be very long but you only want a specific part so the primer tells it where to start
what is the third stage of PCR and explain it?
synthesis - occurs at 72 degrees
- free nucleotides pair up with the exposed bases after the primer
- tag polymerase joins the backbone and forms phosphodiester bonds (instead of human polymerase)
- forms two new strands of DNA
in the third stage of PCR what type of polymerase is used?
tag polymerase which comes from bacteriai
in PCR why is tag polymerase used instead of normal human polymerase?
it can withstand the high temps of 95 degrees without denaturing
who developed DNA sequencing?
sanger
what is DNA sequencing?
a technique used to map out an individuals genome
what ingredients do you need for DNA sequencing?
- DNA sample to be sequenced
- free nucleotides
- coloured fluorescent terminator bases
- DNA primers
- DNA polymerase
in DNA sequencing how does the size of the fragment affect how far it travels in electrophoresis?
shorter fragments travel further, than longer fragments which travel short distances
in DNA sequencing how do we know the order of the fragments from the electrophoresis plate?
the one that have travelled the further are the shortest so would have been at the beginning of the DNA sequence so you can determine the order of fragments
what are the steps in DNA sequencing?
- starts off similar to PCR
- heat the DNA double helix to 95 degress which breaks the H bonds
- then you have two single strands
- then cool to 50 degrees to allow to primer to anneal
- heat to 60 degrees to allow the free nucleotides to anneal to the complementary bases
- but you have terminator bases that stop any more bases being added to the strand
- so you produce lots of fragments of DNA of different lengths
- then you can use gel electrophoresis to analyse this and find the order to the fragments
- can also use lasers over the gel plate to show the order of the fragments
in DNA sequencing what is the role of the terminator bases?
due to the fact they have one less oxygen this means they cannot form phosphodiester bonds so stop the addition of any more bases
what is southern blotting using electrophoresis?
using radioactive DNA probes and xrays
what are the two ways to see the DNA fragments on the electrophoresis plate?
- using southern blotting using radioactive DNA probes and xrays
- fluorescent dyes, DNA probes and UV light
what are the two ways to isolate the desired gene in the first stage of genetic engineering?
using restriction endonucleases:
- cut a specific part of the genome from the restriction sites
- cuts them in a zigzag shape to produce sticky ends so that they are bases exposed which allows it to bind to the plasmid more easily
using reverse transcriptase:
- allow a cell to make mRNA and then extract the mRNA
- using reverse transcriptase, to revert the mRNA back to DNA
- this is called cDNA
what is a vector?
this carries the desired gene
what is the second stage of genetic engineering (after the desired gene has been isolated)?
- extract the plasmid from the bacterium
- cut the plasmid using the same restriction endonucleases so that the same sticky ends are produced
- this allows the desired gene to then attach to the plasmid due to sticky ends at the marker gene
- DNA ligase forms the phosphodiester bonds between the desired gene and the plasmid
- forms recombinant DNA
what is the differnece between DNA polymerase and DNA ligase?
DNA polymerase is used to join nucleotides together in DNA replication.
DNA ligase is used to join different DNA fragments together
what is the recombinant DNA?
new DNA that contains a gene from something else
what are three methods of stage 3 of genetic engineering to transform the cell?
- adding the bacterium and plasmid to Ca+2 rich solution and using heat shock to make the plasma membrane more permeable
- electroporation: uses electricity to open up the pores of the membrane more so it is more permeable
- electrofusion: plasmid is added into a vesicle and electric current is applied to both the plasmid and bacteria so they eventually fuse together allowing the plasmid to move into the bacterium
- creates the transgenic bacteria
what is meant by transgenic bacteria?
a bacterium cell with DNA/genes from another organism
in genetic engineering, when the transgenic bacteria is formed from electroporation, what is the last step?
mass production and culturing by adding the bacteria to an agar plate with nutrients and ampicillin allowing it to grow
after genetic engineering, what are the possible types of product and what are the fates of them all?
bacteria cell with no plasmid:
- has no ampicillin marker gene so dies therefore does not show on plate
bacteria cell with normal plasmid:
- can survive and turns blue as it has the other marker gene
bacteria cell with the genetically modified plasmid:
- can survive as it has the ampicillin marker but has an interrupted blue marker so will be a different colour
what is a use of genetic engineering?
to produce human insulin as they used to use pig insulin but this has issues (rejection, religion, infection)
what is the role of the marker genes?
allows the scientists to be able to tell the difference between plasmids that have been successfully genetically engineered
what is gene therapy?
the process by which a genetic disease can be cured by masking the effect of a faulty allele through the insertion of a healthy one
what are the ethical issues with gene therapy?
- could affect other genes
- replacing non functioning allele could lead to designer babies
what are designer babies?
could lead to non desirable alleles being replaced by desirable alleles in the early embryo
