A2 OCR Biology - Unit 2, Module 2 - Genetic Manipulations

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  • Created by: Jess
  • Created on: 11-04-14 14:51
What does PCR stand for?
Polymerase chain reaction
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What does PCR do?
Makes millions of copies of a fragment of DNA in just a few hours
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How many times is the temperature changed during PCR?
4 times
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What are the 4 different temperatures that PCR undergoes?
95 - 65 - 72 - 95
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What is in the PCR starting mixture?
The DNA sample, free nucleotides, primers, DNA polymerase
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What are primers?
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
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Why is the DNA mixture 1st heated to 95 degrees C (during PCR)?
To break the hydrogen bonds between the 2 strands of DNA
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Why is the DNA mixture secondly cooled to 65 degrees C (during PCR)?
So that the primers can anneal (bind) to the strands
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Why is the DNA mixture thirdly heated to 72 degrees C (during PCR)?
So that DNA polymerase can work
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What does DNA polymerase do during PCR?
Lines up free DNA nucleotides alongside each template strand - for complementary base pairing rules
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In 1 cycle of PCR, how many copies of the DNA fragment are formed?
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Why can PCR be regarded as an exponential cycle?
Each PCR cycle doubles the amount of DNA produced - after the 1st cycle, 2 copies are made, then both of these copies with the 2 original strands are used as templates in the second stage to make 8 fragments....and so on
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How do you get a DNA fragment from an organism's DNA?
Use restriction enzymes
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What does it mean if a section of DNA has a palindromic sequence of nucleotides?
Antiparallel base pairs read the same in opposite directions
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Why do restriction enzymes work?
They recognise specific palindromic sequences and can cut the DNA at these places
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What is the name of the reaction that occurs when restriction enzymes cut the DNA fragment?
Hydrolysis reaction
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Definition of sticky ends
Small tails of unpaired bases at each end of the fragment - used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
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What is added to all DNA fragments so they can be viewed under UV light?
A fluorescent tag
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Why is a buffer solution added during electrophoresis?
It conducts electricity
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What charge do DNA fragments have?
Negative charge
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Which end do the DNA fragments move to during electrophoresis?
The positive electrode at the far end of the gel - the anode
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How is the size of a DNA fragment measured?
In bases
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Why do DNA fragments separate according to size during electrophoresis?
Small DNA fragments move faster and travel further through the gel
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What are organisms that have had their DNA altered by genetic engineering called?
Transformed organisms or genetically modified organisms
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What is an organism that has been genetically engineered to include a gene from a different species called?
A transgenic organism
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What are the 4 stages of genetically engineering a microorganism?
Obtain desired gene in DNA fragment, insert fragment into vector, transfer gene into bacteria, identify transformed bacteria
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Definition of a vector
Something that's used to transfer DNA into a cell - plasmids or bacteriophages (viruses that infect bacteria)
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What are plasmids?
Small, circular molecules of DNA in bacteria
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Why is the same restriction enzyme used to cut the DNA fragment and to cut open the vector DNA?
So the sticky ends of the vector will be complementary to the sticky ends of the DNA fragment containing the gene
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What is the process of ligation?
When the vector DNA and DNA fragment are mixed with DNA ligase and then the DNA ligase joins up the sugar-phosphate backbones of the 2 bits
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What is the new combination of bases in the DNA called after DNA ligase has sealed the vector DNA and DNA fragment during the genetic engineering of a microorganism?
Recombinant DNA
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What are the 2 types of vectors used during genetic engineering?
Plasmid or bacteriophage
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How is a bacterial cell persuaded to take in the plasmid vector?
Bacterial cells placed into ice-cold calcium chloride solution to make their cell walls more permeable, plasmids added, mixture is heat-shocked to around 42 degrees C for 2minutes
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How does a bacteriophage vector make the bacterial cell take up the recombinant DNA?
Bacteriophage infects bacterium by injecting its DNA into it - the phage DNA then integrates into the bacterial DNA
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What are the cells that takeup the vectors containing the desired gene called?
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Why do you need to identify the transformed bacteria after they have taken up a vector?
Because not all of the bacteria will have taken up the vector
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How can you identify the transformed bacteria after they have taken up a vector?
Insert marker genes into the vectors at the same time as you insert the desired gene
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Why is it useful for microorganisms to take up plasmids?
The plasmids often contain useful genes so microorganisms can gain useful charateristics and have an advantage over other microorganisms, increasing their chances of survival
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Name 3 genes that plasmds may contain
Genes that code for antibiotic resistance, genes that help microorganisms invade hosts (breaking down tissues), genes that mean microorganisms can use different nutrients (e.g break down sugars that aren't normally used)
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Describe the process of transforming bacteria to produce human insulin
1. Identify and isolate gene for human insulin using restriction enzymes/ 2. Cut open plasmid (same r. enzymes)/ 3. Insert insulin gene into plasmid/ 4. Plasmid taken up by bacteria/ 5. Bacteria identified using marker genes and grown in fermentor
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What do you have to do to the human insulin created by bacteria once it's been made?
Extract and purify it
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Why is genetically engineered human insulin more effective over animal insulin?
Identical - more effective/less risk of allergic reaction, cheaper and faster to produce, more reliable and larger supply produced, overcomes ethical/religious issues arising from using animal insulin (Jewish)
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How does Golden Rice produce beta-carotene?
Genetically engineered to contain a gene from a daffodil plant and a gene from a soil bacterium
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What is beta-carotene used for in our bodies?
To produce Vitamin A
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What are the names of the 2 genes inserted into Golden Rice from a daffodil and soil bacterium?
psy and crtl (respectively)
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Describe the process of inserting plasmid into rice cells (1st stages)
1. Isolate psy and crtl genes with r. enzymes/ 2. Remove plasmid from bacteria (same r. enzymes)/ 3. Insert psy, crtl genes and a marker gene into plasmid/ 4. Put plasmid back into bacteria/ 5. Incubate rice cells with transformed bacteria
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Describe the process of turning this rice into Golden Rice (last stages)
6. Bacteria infect rice plant cells/ 7. Bacteria inserts genes into rice's DNA/ 8. Transformed rice/ 9. Rice grown on a selective medium - only rice with the marker gene (and therefore the gene for beta-carotene) will be able to grow
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What is the Latin name of the bacteria that infects rice plants?
Agrobacterium (A.) tumefaciens (in Italics)
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What is xenotransplantation?
The transfer of cells, tissues or organs from one species to another
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Is there a greater risk of rejection with xenotransplantation?
Yes - because of the genetic differences between organisms of different species are greater than between organisms of the same species
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How can an organ be rejected?
The immune system recognises proteins on the surface of the transplanted cells as foreign and starts an immune response against them
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What are scientists trying to do so the organs of animals aren't rejected when transplanted into humans?
Genetically engineer the organs
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What is the 1st way that scientists are genetically engineering the organs of animals so they aren't rejected when transplanted into humans?
Genes for human cell-surface proteins are inserted into a newly fertilised animal embryo so the genes integrate into the animal's DNA. The animal then produces human cell-surface proteins
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What is the 2nd way that scientists are genetically engineering the organs of animals so they aren't rejected when transplanted into humans?
Remove or inactivate animals genes in the nucleus that are involved in making cell-sruface proteins, transfer the nucleus to an unfertilised animal egg cell which is then stimulated to divide into an embryo. The animal produced won't produce animal c
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Describe an example of how scientists have been able to remove or inactive a gene in an animal that is involved in making cell-surface proteins
Pigs have a sugar Gal-alpha (1,3)-Gal on their cell surface proteins which humans don't - scientists have developed a pig that doesn't produce the enzyme needed to make this sugar
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Describe some ethical issue surrounding genetic engineering
Increased number of antibiotic-resistant pathogenic microorganisms, GM crops may encourage monoculture which decreases biodiversity, animals may suffer from xenotransplantation, superweeds,biotechnology companies may exploit farmers in poor countries
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What does gene therapy involve?
Altering alleles inside cells to cure genetic disorders
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To use gene therapy on a disease caused by 2 recessive alleles you must...
Add a working dominant allele to make up for them
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To use gene therapy on a disease caused by a dominant allele you must...
Silence the dominant allele - e.g. by sticking a bit of DNA in the middle of the allele so it doesn't work any more
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Name 3 different types of vectors that can be used for gene therapy
Altered viruses, plasmids or liposomes
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How do you get the new allele inside the cell during gene therapy?
Insert the allele into the cell using vectors
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Name the 2 types of gene therapy
Somatic and germ line
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Describe somatic gene therapy
Alter the alleles in body cells (those most affected by the disease) - offspring could still inherit the disease
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Which gene therapy (somatic or germ line) is currently illegal?
Germ line
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Describe germ line therapy
Alter the alleles in the sex cells - so every cell of any offspring will not suffer from the disease
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Which gene therapy (somatic or germ line) is used to treat cystic fibrosis?
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What are the advantages of gene therapy?
Prolong life, better quality of life, carriers may be able to have kids without the disease (GL only), decrease number of people suffering from genetic disorders
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What are the disadvantages of gene therapy?
Expensive, may be a short-lived treatment that has to be undergone multiple times (S), difficult to get allele into specific cell, immune response activated against the "foreign" body cells, allele inserted into wrong place
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What are DNA probes?
Short strands of DNA with a specific base sequence that's complementary to the target sequence that you are looking for
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What are DNA probes used for?
Identifying DNA fragments that contain specific sequences of bases
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What happens if your DNA probe locates the specific sequence of bases?
It will bind (hybridise) to the target sequence
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What are the 2 types of label that can be attached to a DNA probe?
A fluorescent label (detected under UV light) or a radioactive label (detected using X-ray film)
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Give a use of DNA probes
Seeing if any members of a family has a mutation in a gene that causes a genetic disorder
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Describe how DNA probes are used to see if any members of a family has a mutation in a gene that causes a genetic disorder
1. Digest DNA samples into fragments using r. enzymes/ 2. Separate fragments using electrophoresis/ 3. Transfer to nylon membrane/ 4. Incubate with fluorescent DNA probe/ 5. Probe will bind to gene if it's present/ 6. UV light exposes gene
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How can you sequence a whole genome?
Using BACs (bacterial artificial chromosomes)
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What are BACs?
Bacterial artificial chromosomes - man-made plasmids
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What is the chain-termination method used for?
To sequence the order of bases in a section of DNA
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What is in the mixture used in the chain-termination method?
Single-stranded DNA template to be sequenced, DNA primers, DNA polymerase, free nucleotides (A, T, C and G's) and flurorescently-labelled modified nucleotides (A*, T*, C* and G*)
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How many tubes is the mixture used in the chain-termination method put into and why?
4 separate tubes - one with A*, C*, G* or T*
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Describe the chain-termination method
1. Add mixture to tubes/ 2. PCR produces many DNA strands of different sizes (stand ends with addition of modified nucleotide)/ 3. Electrophoresis separates fragments by size/ 4. UC light shows fluorescent labels/ 5. Read base sequence on gel
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Where is the smallest nucleotide on the gel during the chain-termination method?
At the bottom of the gel
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How many base pairs long can the chain-termination method be used for?
750 bp long
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Why can't you use the chain-termination method on a whole genome?
The chain-termination method can only be used for DNA of 750 bp long so the genome needs to be cut into smaller pieces first
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In one sentence, describe how a whole genome can be sequenced
Genome cut into fragments, the smaller ones of which are sequenced then put back into order
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When the genome is cut using r. enzymes, what size are the smaller fragments?
100 000 bp
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Describe how a genome can be sequenced
1. Cut genome with r.e.'s/ 2. Insert each fragment into a different BAC/ 3. BACs inserted into bacteria - divide into colonies/ 4. DNA extracted from each colony and cut up with r.e.'s (overlaps)/ 5.Sequenced by chain-termination method and computers
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What is the role of computers when sequencing a whole genome?
They put the DNA fragments from all the BACs into order
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Why compare genomes of different species?
Understand evolutionary relationships - see how closely related they are, understand how genes interact/are controlled during development, carry out medical research (human diseases found in other animals)
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Why compare genomes of the same species?
Trace early human migration, study the genetics of human diseases, develop medical treatments specific to particular genotypes (medicine may be more effective in 1 person than another)
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What does PCR do?


Makes millions of copies of a fragment of DNA in just a few hours

Card 3


How many times is the temperature changed during PCR?


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Card 4


What are the 4 different temperatures that PCR undergoes?


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Card 5


What is in the PCR starting mixture?


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