Biology last CAT- teacher B Flashcards
What is a genome?
the entire set of DNA, including all the genes in an organism.
What is a proteome?
all the proteins that are made by an organism genome.
What makes it more difficult to translate a genome into the proteome?
- more complex organisms contain large sections of non coding DNA
- they also contain complex regulatory gene which determine when the genes that code for specific proteins should be turned on or off.
What is recombinant DNA technology?
- involves the transferring of a fragment on DNA from one organism to another.
- because the genetic code is universal and because transcription and translation mechanisms are similar the the transformed DNA can be used to produce a protein in the cells of the recipient organism.
- The recipient and donor don’t even have to be from the species.
Describe how DNA fragments can be made using reverse transcriptase:
1) most cells only contain two copies of each gene, making it difficult to obtain a DNA fragment containing the target gene.But they may contain many mRNA molecules which are complementary to the gene, so mRNA is often easier to obtain.
2) The mRNA molecules can be used as templates to make lots of DNA.The enzyme, reverse transcriptase, makes DNA from an RNA template.The DNA produced is called complementary DNA.
3) For example, pancreatic cells produce the protein insulin.They have lots of mRNA molecules complementary to the insulin gene, but only two copies of the gene itself.So reverse transcriptase could be used to make cDNA from the insulin mRNA.
4) To do this mRNA is first isolated from cells.Then its mixed with free DNA nucleotides and reverse transcriptase.The reverse transcriptase then uses the mRNA as a template to synthesise a new strand of cDNA.
Describe how DNA fragments can be made using restriction endonuclease enzymes:
1) Some sections of DNA have palindromic sequences of nucleotides.These sequences consist of antiparallel base pairs (base pairs that read the same in the opposite direction)
2) Restriction endonuclease are enzymes that recognise specific palindromic sequences and cut the DNA at these places.
3) Different restriction endonucleases cut at different specific recognition sequences,becuase the shape of the recognition sequence is complementary to the active site of the enzyme.
4) If recognition sequences are present at either side of the DNA fragment you want you can use can use restriction endonucleases to separate it from the rest of the DNA.
5) The DNA sample is then incubated with the specific restriction endonuclease, which cuts the DNA fragment out via a hydrolysis reaction.
6) Sometimes the cut leaves sticky ends- small tails of unpaired bases at each end of the fragment.Sticky ends can be used to bind the DNA fragment to another piece of DNA that has sticky ends with a complementary sequences.
Describe how DNA fragments are made using a gene machine:
1) More recently, technology has been developed so that fragments of DNA can be synthesised from scratch, without the need for preexisting DNA templates.
2) Instead a database contains the necessary information to produce the DNA fragment.
3) This means that the DNA sequence does not have to exist naturally- any sequence can be made.
4) How its done:
- The sequence that is required is designed if it does already exist.
- The first nucleotide in the sequence is fixed to a support e.g a bead
- nucelotides are added step by step in the right order , in a cycle of processes that includes adding protecting groups.Protecting groups make sure that the nucleotides are joined at the right points to prevent unwanted branching.
- Short sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced.Once these are complete, they are broken off from the support and all the protecting groups are removed.The oligonucleotides can then be joined together to make longer DNA fragments.
Describe in vivo amplification:
STEP 1- THE DNA FRAGMENT IS INSERTED INTO A VECTOR:
1)a vector is something that’s used to transfer DNA into a cell.They can be plasmids or bacteriophages.
2)The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene.So the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene.
3)The vector DNA and DNA fragment are mixed together with DNA ligase.DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA.This process is called ligation.
4)The new combination of bases in the DNA is called recombinant DNA.
STEP 2- THE VECTOR TRANSFERS THE DNA FRAGMENT INTO HOST CELLS:
1)The vector with the recombinant DNA is used to transfer the gene into cells.
2)If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
3)With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it.The phage DNA then integrates into the bacterial DNA.
4)Host cells that take up the vectors containing the gene of interest are said to be transformed.
STEP 3- IDENTIFYING TRANSFORMED HOST CELLS:
only 5% of host cells are transformed
1)Marker genes can be inserted into vectors at the same time as the gene to be cloned.This means any transformed host cells will contain the gene to be cloned and the marker gene.
2)Host cells are grown on agar plates.Each cell divides and replicates its DNA, creating a colony of clones cells.Transformed cells will produce colonies where all the cells contain the cloned gene and the marker gene.
3)The marker gene can code for antibiotic resistance- host cells are grown on agar plates containing the specific antibiotic, so only transformed cells that have the marker gene will survive and grow.Or it can code for fluorescence- when the air plate is put under UV light only transformed cells will fluoresce.
4)Identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene.
How to make transformed cells produce proteins?
1) make sure the vector contains specific promotor and terminator regions.
2) Promotor regions are DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA.Terminator regions tell it when to stop.Without the right promotor region, the DNA fragment won’t be transcribed by the host cell and a protein won’t be made.
3) Promotor regions may be present in the vector DNA or they may have to be added in along with the fragment.
Describe how DNA fragments can be amplified in vitro amplification?
1) A ruction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
- primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
- DNA polymerase is an enzyme that creates new DNA strands.
2) The DNA mixture is heated to 95 degrees to break the hydrogen bonds between the two strands of DNA.
3) the mixture is then cooled to between 50 and 65 degrees so that primers can bind to the strands.
4) the reaction is heated to 72 degrees so DNA polymerase can work.
5) The DNA polymerase lines up free DNA nucleotides alongside each template strand.Specific base pairing means new complementary strands are formed.
6) Two new copies of the fragment of DNA are formed and on cycle of PCR is complete.
7) The cycle starts again, with the mixture being heated to 95 degrees and this time all four strands are used as templates.
8) Each PCR cycle doubles the amount of DNA.
Describe how gene therapy could be used to cure genetic disorders and cancer:
1) Gene therapy involves altering the defective genes inside cells to treat genetic disorders and cancer.
2) How you do this depends on whether the disorder is caused by a mutated dominant allele or two mutated recessive alleles
- If its caused by two mutated recessive alleles you can add one working dominant allele.
- If its caused by a mutated dominant allele you can silence the dominant allele e.g by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore/
3) The allele is inserted into cells using vectors just like in recombinant DNA technology
4) Different vectors can be used e.g altered viruses, plasmids or liposomes
5) There are two types of gene therapy:
- Somatic therapy- involves altering the alleles in body cells particularly the cells that are most affected by the disorder.For example cystic fibrosis the epithelial cells lining the lungs.Somatic therapy doesn’t affect the individuals sex cells so offspring could still inherit the disorder.
- Germ line therapy- involves 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 suffer the disease.Germ line therapy in humans is currently illegal.
Describe how you can use DNA probes and hybridisation to look for alleles?
1) DNA probes can be used to locate specific alleles of genes or to see if a persons DNA contains a mutated allele that causes a genetic disorder.
2) DNA probes are short strands of DNA.They have a specific bas sequence that’s complementary to the base sequence of part of a target allele.
3) This means a DNA probe will bind to the target allele if it’s preset in a sample of DNA.
4) A DNA probe has a label attached, so that it can be detected.The two most common types of label are a radioactive label detected using X-ray film or a fluorescent label.
5) Heres how it is done:
- A sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresis.
- The separated DNA fragments are then transferred to a nylon membrane and incubated with the fluorescent labelled DNA probe.
- If the allele is present, the DNA probe will hybridise to it.
- The membrane is then exposed to UV light and if the gene is present there will be a fluorescent band.
6) Alternatively the probe can be used as part of DNA microarray, which can screen lots of genes at the same time:
- A DNA microarray is a glass slide with microscopic spots of DNA probes attached to it in rows.
- A sample of fluorescently labelled human DNA is washed over the array.
- If the labelled human DNA contains any DNA sequences that match any of the probes, it will stick to the array.
- The array is washed to remove any labelled DNA that hasn’t stuck to it.
- The array is then placed under UV light, any spot that fluoresces means that the persons DNA contains that specific allele.
Describe genetic fingerprinting:
1) A sample of DNA is obtained
2) PCR is used to make many copies of the areas of DNA that contain the VNTRs- primers are used that bind to either side of these repeats so the whole repeat is amplified.
3) You end up with DNA fragments where the length corresponds to the number of repeats the person has at each specific position.
4) A fluorescent tag is added to all the DNA fragments so they can be viewed under UV light.
5) The DNA fragments undergo electrophoresis:
- 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- DNA fragments are negatively charged, so they move towards the positive electrode at the far end of the gel.
- Small DNA fragments move faster and travel further through the gel, so the DNA fragments separate according to size.
6) The DNA fragments are viewed as bands under UV light this is the genetic fingerprinting.
7) Two genetic fingerprints can be compared.
Biodiversity
The variety of living organisms in an area.
Habitat
the place where an organism lives.
