8B Genome Projects and Gene Technologies Flashcards
Genome
The entire set of DNA
Proteome
All the proteins that are made by an organism
Why is it easy to determine the proteome of a simple organism?
They don’t have any introns that have to be removed
Recombinant DNA
The name for DNA formed by joining together DNA from different organisms
What are the three methods of making DNA fragments?
Use reverse transcriptase. use restriction endonuclease, use a gene machine
How is reverse transcriptase used to make a DNA fragment?
The mRNA molecules are used as template, and reverse transcriptase makes DNA from this mRNA template.
mRNA is mixed with free DNA nucleotides and reverse transcriptase. These make single strands of cDNA
What is the DNA that is produced by using mRNA and reverse transcriptase called?
cDNA (complementary DNA)
Why is mRNA used with reverse transcriptase?
There’s only two copies of each gene, so it is difficult to obtain a DNA fragment from the actual DNA. There are lots of copies of mRNA that are used to code for proteins, so mRNA is easier to obtain
How are restriction endonuclease enzymes used to make a DNA fragment
Restriction endonuclease enzymes recognise specific palindromic sequences and cut the DNA at these sequences, because the shape of the recognition sequence is complementary to the enzymes active site
Palindromic sequences
Sequence that consist of antiparallel base pairs (base pairs that read the same in opposite directions)
How can DNA fragments cut with restriction endonuclease enzymes be joined together?
If cut with the same restriction endonuclease, it will create sticky ends. The complementary sticky ends of the DNA fragments can anneal
Sticky end
A small tail of unpaired DNA bases at the end of a DNA fragment
How are DNA fragments made using a gene machine?
DNA synthesised from scratch. Sequence that is needed is designed on the computer. The first nucleotide is fixed to a support e.g a bead. Nucleotides are added. Oligonucleotides (twenty nucleotides long). Oligonucleotides are joined together
What are the two ways of amplifying DNA fragments?
In vivo and in vitro
What is the first step in in vivo cloning?
To insert the DNA fragment into a vector’s DNA
vector
Something used to transfer DNA into a cell (e.g a plasmid)
Describe the process of in vivo cloning
The vector is isolated
The vector is cut open using the same restriction endonuclease that was used to isolate the DNA fragment of the target gene
Vector and DNA fragment are mixed together with DNA ligase
The vector DNA and the DNA fragment have now been added together, and are a recombinant DNA
What is the purpose of DNA ligase?
To anneal the sticky ends together
Ligation
The use of DNA ligase to anneal the sticky ends
Transforming cells
Inserting the vector with the recombinant DNA into a cell
How do you get cells to take up the recombinant DNA?
Place bacterial cells in an ice cold calcium chloride solution to make the cell walls more permeable. Then heat shock (42 degrees c for 1-2 mins)
How do you identify transformed cells?
Use marker genes.
Insert marker gene at same time as inserting the DNA fragment into the vector DNA
Host cells grown on agar jelly. Shows evidence of marker gene also such as antibiotic resistance or fluorescence
If you want the transformed cells to produce proteins what do you have to make sure?
The vector contains specific promoter and terminator regions
Promoter regions
DNA sequences that tell the enzyme RNA polymerase where to start producing mRNA
Terminator regions
DNA sequences that tell the enzyme RNA polymerase where to stop producing mRNA
What process is used in in vitro cloning?
PCR (Polymerase chain reaction)
In PCR what does the primary reaction mixture contain?
The DNA sample, free nucleotides, primers and DNA polymerase
Primers
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
Describe the process of PCR
Reaction mixture containing sample DNA, primers, DNA polymerase and free nucleotides. Mixture is heated to 95 degrees c and then cooled to between 50 and 60 degrees c. The reaction mixture is then heated to 72 degrees c. DNA polymerase lines up the free nucleotides along the template strand and creates phophodiester bonds between the nucleotides
What is the result of a single cycle of PCR?
The amount of DNA fragments are doubled
Why is the reaction mixture in PCR heated to 95 degrees c?
To break the hydrogen bonds between the two strands of DNA
Why is the reaction mixture in PCR cooled to 50-60 degrees c?
So the primers can anneal to the strands
Why is the reaction mixtures in PCR heated to 72 degrees c?
So DNA polymerase can work, and form complementary strands of DNA to the template strand
What are the two types of gene therapy?
Somatic therapy and germ line therapy
What is somatic therapy?
Altering the alleles in the body cells. Doesn’t alter the sex cells, so offspring could still inherit the disease
What is germ line therapy?
Altering the alleles in the sex cells so any offspring produced from these cells will be affected by the germ therapy and they won’t suffer from the disease.
What are the benefits of using transformed organisms in agriculture?
Crops could give higher yields, or more nutritious yields, therefore reducing the risk of famine or malnutrition. Can also make them resistant to pests or droughts
What are the benefits of using transformed organisms in industry?
The production of enzymes in large quantities, therefore reducing the cost
What are the benefits of using transformed organisms in medicine?
Produce drugs or vaccines cheaply or in large quantities, so it makes them more available and affordable to people who need them
What are the concerns of using transformed organisms in agriculture?
Might produce a monoculture, reducing the biodiversity, so if a disease occurs it will kill all crops as non offer an advantageous mutation of resistance to that disease. Could produce a “superweed”. Crops from organic famers can have their crops contaminated by wind blown seeds, therefore they would lose money
What are the concerns of using transformed organisms in industry?
People won’t be able to chose if they consume genetically engineered food. Could create a monopoly of large biotechnology companies
What are the concerns of using transformed organisms in medicine?
Companies may limit the technology that could save lives, or promote the unethical use of genetic engineering, e.g designer babies
How do you use gene therapy to cure a disease caused by two recessive alleles?
Insert a dominant allele
How do you use gene therapy to cure a disease caused by a mutated dominant allele?
“Silence” the dominant allele by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore
DNA probes
Short strands of DNA that are used to locate complementary alleles (the probe has a complementary base sequence to the target allele). This means the probe will bind to the target allele if it is present in a sample of DNA. The probes have a label attached so it can be detected
How are fluorescently labelled probes used to locate a target allele?
A sample of DNA is cut into fragments via a restriction endonuclease and then separated using electrophoresis. The separated DNA fragments are then transferred to a nylon membrane and incubated with a fluorescently labelled DNA probe. If the allele is present, the DNA probe will hybridise to it. The membrane is the exposed to UV light, fluorescence will indicate that the target allele is present
Microarray
A glass slide with microscopic spots of different DNA probes attached to it in rows
What is a microarray used for?
To test for lots of different genes at the same time
How is a microarray used?
Sample of fluorescently labelled DNA is washed over the array. If any of the labelled DNA contains complementary sequences to the probes it will stick to the array. The array is washed, to remove any fluorescence that hasn’t bound. UV light is then used to look at which probes have hybridised, showing which alleles are present
What is screening using DNA probes used for?
To test for an inherited disease, how a patient will respond to drugs, identify health risks, to personalise medicine
Genetic counselling
Advising patients and their relatives about the risks of genetic disorders
VNTRs
Variable Tandem Repeats- base sequences that don’t code for proteins, and repeat. The number of times the VNTR repeats differs from person to person, so the length of these sequences differ
What does genetic finger printing use?
The VNTRs
What is the difference between individuals VNTRs?
The number of repeats (length) and position
What are the three steps of genetic fingerprinting?
Use PCR to make the DNA fragments, Separate the fragments by gel electrophoresis, Analysis of the genetic fingerprints
How is a sample of a persons DNA obtained?
From their blood or saliva
How is gel electrophoresis used to separate the DNA fragments?
The DNA mixture is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity. An electrical current is passed through the gel and the negatively charged DNA fragments move towards the positive electrode. Short DNA fragments more faster and further down the gel, longer fragments move slower and not as far down the gel. Then view using UV light
What are the five uses of genetic finger printing
Determining genetic relationships, determining genetic variability within a population, in forensic science, for medical diagnosis, in animal and plant breeding
