Manipulating genomes Module 6 Flashcards
3 techniques to study genes?
The polymerase chain reaction (PCR)
Gel electrophoresis
Cutting out DNA fragments, using restriction enzymes
How can multiple copies of a DNA fragment be made, using PCR?
A reaction mixture is set up, containing the DNA sample, free nucleotides, primers and DNA polymerase
DNA mixture is heated to 90 degrees, to break the hydrogen bonds between the 2 strands of DNA
Mixture is then cooled to between 50 and 65 degrees, so the primers can anneal (bind) to the strands
Reaction mixture heated to 72 degrees so DNA polymerase can work
DNA polymerase lines up free nucleotides with alongside each template strand, complementary base pairing means new complementary strands are formed
2 new copies of the fragment of DNA are formed, and one cycle of PCR is formed
What are primers?
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
What’s DNA polymerase?
An enzyme that creates new DNA strands
Why is it useful that DNA polymerase doesn’t denature at high temperatures?
Useful as cycles of PCR can be carried out without having to use new enzyme each time
What happens when PCR is repeated?
Now have 4 strands (2 new and 2 original)
So next cycle there will be 8 then 16 then 32…
What is electrophoresis?
Procedure that uses an electrical current to separate out DNA fragments, RNA fragments, or proteins depending on their size
Explain electrophoresis steps
Row of wells is created at the end of some agrose gel, which is in a gel box/tank, with the wells nearest to the negative electrode
Add loading dye to each DNA sample, helps the sample sink to bottom of wells making them easier to see. Add a set amount of each DNA fragment solution to each well
Pass an electrical current through the gel, DNA fragments are negatively charged so will move towards the positive electrode.
Small DNA fragments will move faster and travel further, through the gel causing them to separate by size, turn power off when 2cm from end. Stain the DNA fragments
The bands of different DNA fragments will now be visible
How is electropheresis carried out on proteins?
Proteins can be positively or negatively charged, so before put in wells are mixed with a chemical that denatures the proteins so they all have the same charge
Used to identify blood or urine samples for disease
How can restriction enzymes be used to cut out DNA fragements?
Some sections of DNA have palindromic sequences of nucleotides, these sequences consist of anti-parallel base pairs (base pairs that read the same in the opposite direction)
Restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
Different restriction enzymes cut at different specific recognition sequences because the shape of the recognition sequence is complementary to an enzyme’s active site.
If recognition sequences are present at either side of the DNA you want, you can use restriction enzymes to separate it from the rest of the DNA
DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment via a hydrolysis reaction
Sometimes cut leaves sticky ends (small tails of unpaired bases at each end of the fragment), as cut in a zig zag motion. Sticky ends can be used to anneal the DNA fragment to another piece of DNA, that has sticky ends with complementary sequences
What’s a genome?
All the genetic material in an organism
How can DNA profiles be created, and how do they distinguish between 2 different people?
Some of an organism’s genome contains repetitive non-coding base sequences
The number of times these repeat differ in person to person, therefore their length in nucleotides does aswell
The number of times a sequence is repeated (therefore the amount of nucleotides) at different loci (specific place), can be analysed using electrophoresis. Creating a DNA profile
Why is the probability having the same DNA profile very low?
Because having the same number of sequence repeats at each locus is very low
How can DNA profiling be used in forensic sciences?
DNA taken from all possible suspects
DNA is isolated
PCR is used to amplify multiple areas containing different sequence repeats
PCR products are run on electropheresis gel, and the profiles are compared to see if any match (have the same bands)
How DNA profilling be used to identify to see if samples have come from the same species?
The more similar the bands, the more likely they are from the same species
How can DNA profilling be used in medical diagnosis?
A DNA profile can refer to a unique pattern of several alleles
It can be used to analyse the risk of genetic disorders, where specific mutation is unknown or several mutations may have occurred, because it identifies a broader, altered genetic pattern
What is genetic engineering?
The manipulation of an Organism’s DNA
What’s a transformed organism?
Organisms that have had their DNA altered by genetic engineering, so have recombinant DNA (DNA formed by joining together DNA from different sources)
Brief outline of how genetic engineering process?
Involves extracting a gene from one organism, and then inserting it into another organism, normally of a different species. Or genes can also be manufactured instead of extracted from another organism.
The organism with the inserted gene will now produce the protein coded for by that gene
In depth description of how first step of genetic engineering works?
DNA fragment containing the desired genes is isolated using restriction enzymes
In depth explanation how the second step of Genetic engineering works?
The DNA fragment is inserted into a vector DNA (Vector is something used to transfer DNA into a cell, eg. plasmids (small circular molecules of DNA in bacteria, or bacteriophages (viruses that infect bacteria))
The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment that containing the desired gene. So the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene
The vector DNA and DNA fragment are mixed together with DNA ligase
DNA ligase joins up the sugar-phosphate backbone of the 2 bits, = ligation
In depth explanation how the third step of genetic engineering works?
The vector with the recombinant DNA is then used to transfer the gene into the bacterial cells
If a plasmid vector is used, bacterial cells have to be convinced to take in the plasmid vector, and it’s DNA
Eg. suspension of bacterial cells is mixed with plasmid vector in a machine called an electroporator
The machine is switched on and an electrical field is created in the mixture, increasing the permeability of the bacterial cell membranes, and allows them to take in the plasmids, this is called electroporation
With a bacteriophage plasmid, the bacteriophage will infect the bacterium by injecting it’s DNA into it, the phage DNA then intergrates with the bacterial DNA
How can genetic engineering be used to improve plant yield?
Inserted with a gene that produces a protein that is toxic to some insects
Also positive ethical issue, as less chemical pesticides that farmers have to use on their crops
Negative ethical issues, promotes monoculture (only one type of crop planted, decreasing biodiversity, and leaves all crops vunerable to one disease as all genetically indentical
How can genetic engineering be used on animals?
They can be used to be make proteins, DNA fragments for protein injected into animal embryo, the embryo is then implanted into a female adult, when offspring is born, it’s tested to see if it can produce the protein. If it can it is reproduced using selective breeding
The protein is then extracted from the milk and put in the medicine
Positive ethical with pharming, is that drugs can be made in larger quantities, saving more people
Negative ethical issue, is that can harmful side effects o animals, and makes animals only assests
Example of how genetic engineering be used be carried out on pathogens for research?
Tumour cells have receptors on their membranes for the polio virus, so poliovirus will recognise and attack them
So can genetically engineer the poliovirus, to deactivate the genes that cause poli,but it will still attack and kill cancer cells
Ethical pros and cons of genetically altering pathogens?
Positives- new disease can now be cured
Negatives-
scientists doing research become infected and spread the disease
Genetically modified version of pathogen could revert to original form, and cause an outbreak of disease
Could be used in bio-warfare
Ethical pros and cons of companies patterning genetically engineered organisms?
Positives- there’s more incentive to create genetically engineered organisms, as more money so more scientific breakthroughs are made
Negatives- In third world countries, may have to rely on genetically engineered organisms ie crops, but pattern regulations mean they have to pay excessive amounts
What’s gene therapy?
Altering alleles inside cells, to cure genetic disorders
How do you use gene therapy if the problem is caused by 2 recessive alleles?
Add a working dominant allele
How do you use gene therapy if it’s caused by a dominant allele?
You can silence the dominant allele by adding a piece of DNA in the middle so it doesn’t function anymore
How do you get an allele inside a cell?
Using a vector
What’s somatic therapy?
Altering the alleles in body cells, particularity targeting the cells most affected by the disorder
Somatic therapy doesn’t affect the individuals sex cells, so any offspring could inherit the disease
What’s germ line therapy?
Altering the alleles in the sex cells, this means every cell of any offspring produced from these cells will be affected by the gene therapy and they won’t inherit the disease, currently illegal
Positive ethical issues of gene therapy?
Prolong lives and increase quality of life of people with genetic disorder
Germ therapy means people can have children without spreading disease, therefore also reducing the amount of people with the disease
Negative ethical issues of gene therapy?
Could be used not only for medical reasons, but cosmetic reasons
Very expensive, money could be used more wisely
In somatic therapy effects may be short lived, and have to undergo multiple treatments
The body could identify vectors as foreign bodies and start an immune response against them
Allele may be difficult to insert into specific body cell, or entered into wrong place, causing cancer
Inserted allele could be overexpressed, producucing too much of the missing protein
How can DNA be sequenced using the chain-termination method?
The following mixture is added to 4 separate tubes:
A single stranded DNA template
Lots of DNA primers
DNA polymerase
Free nucleotides
Florescently labelled modified nucleotide, like a normal nucleotide, but once it’s added to a DNA strand no more bases can be added after that, (a different modified base is added to each tube A,T,C,G
The tubes undergo PCR which produces many strands of different length DNA, because each one terminates at a different point
The DNA fragments in each tube are separated by electropheresis, and visualised under UV light
The complementary base sequence can be read from the gel as smallest length is first and longest is last
(Have to flip to find sequence of original strand)
How can gene sequencing techniques be used to find whole genomes?
A genome is cut into smaller fragments using restriction enzymes
The fragments are inserted into different bacterial artificial chromosomes (man made plasmids)
The BAC’S are then inserted into bacteria-each bacterium containing a BAC with a different DNA fragment
The bacteria divide, creating colonies of identical cells that all contain a specific DNA fragment, together the different colonies form genomic DNA libary
DNA is extracted from each each colony, and cut up using restriction enzymes producing overlapping pieces of DNA
Each piece of DNA is sequenced using chain-termination method, and put together back in order to give full sequence from BAC
Sequences from all BAC’s put together to form the whole genome
Explain how faster whole genomes sequencing techniques have been developed to improve chain termination technique?
Chain termination technique, is now automated and faster, as each tube contains all the terminator sequences, and instead having a running gel, machine just reads them out
Explain pyrosequencing the faster whole genome sequencing technique
Section of DNA is cut into fragments, split into single strands, and then a strand from each fragment is attached to a small bead
PCR is used to amplify the DNA fragments, and each bead, then each bead is put into a separate well
Free nucleotides are added to the wells and attach to the DNA via complementary base pairing
Wells also contain specific enzymes which cause light to be given off when a certain base attaches
Computers analyse light intesities and allows them to calculate the whole genome
What is synthetic biology?
The construction of new biological systems using DNA made from scratch
How does sequencing genomes aid synthetic biology?
Amino acids are coded for by triplets of bases on a gene
So from the sequence of a gene can predict the primary structure of a protein
How can comparing sequenced genes or whole genomes be useful?
To study genotype/phenotype relationships
In epidermiological studies (study of health and disease within a population)
To help understand evolutionary relationships, more similar genomes = more closely related
Explain pyrosequencing the faster whole genome sequencing technique?
Section of DNA is cut into fragments, split into single strands, and then a strand from each fragment is attached to a small bead
PCR is used to amplify the DNA fragments, and each bead, then each bead is put into a separate well
Free nucleotides are added to the wells and attach to the DNA via complementary base pairing
Wells also contain specific enzymes which cause light to be given off when a certain base attaches
Computers analyse light intesities and allows them to calculate the whole genome
