Genetic Technology Flashcards
Suggest how a gene from one species can be inserted into a bacteria (3)
Gene isolated and cut with a restriction enzyme. The plasmid is cut with the same restriction enzyme to create complimentary sticky ends. Plasmid and gene are mixed with DNA ligase and the plasmid is taken up by the bacteria
How does a marker gene show that the gene has been uptaken (3)
The marker gene is linked to the gene for wanted protein, with the promoter. When the marker gene is expressed you can observe it, for example it fluoresces. You can then tell that the wanted gene is also being transcribed.
Why does the promoter need to be transferred with the desired gene (3)
The promoter initiates transcription. As this is where RNA polymerase binds. Otherwise the gene will have to be inserted near an existing promoter, it is difficult to do this as it disrupts the expression of the existing gene.
Why are fluorescent markers preferred to antibiotic resistance markers (2)
Easier to identify, more economical / time saving. Resistance genes can be passed to other bacteria. The antibiotics will no longer be effective, so there will be no treatment for the disease.
Recombinant DNA
DNA made by joining pieces from two or more different sources
An overview of gene transfer
The gene that is required is identified. It may be cut from a chromosome, made from mRNA by reverse transcription or synthesised from nucleotides. Multiple copies of the gene are made by PCR. The gene is inserted into a vector which delivers the gene to the cells of the organism. The virus takes the gene into the cell. The cells that have the new gene are identified and cloned
Example of vectors
Plasmids, viruses and liposomes
Why do we use PCR (polymerase chain reaction)
A methord of rapidly producing a very large number of copies of a fragment of DNA
Methord of PCR
DNA is heated to denature the DNA, which separates the helix. A primer DNA is used after cooling, complimentary base pairing occurs. DNA polymerase uses free nucleotides to synthesise complimentary strands. The gene has now been copies and forms part of two DNA molecules. This process can be repeated to produce more DNA
Forensis medecine
PCR is used in forensic medicine to amplify DNA from the smallest tissue sample left at the scene of the crime. Electrophoresis can then be used to compare DNA samples.
Taq poymerase
The first heat stable DNA polymerase to be used in PCR. It synthesises complementary DNA strands. It works at high temperatures, so does not need to be replaced after each cycle. Process is more efficient than normal DNA polymerase.
PCR anealing
Attaching the primer to the end of the single stranded DNA molecule 65 degrees
PCR denaturing
Turing the double stranded DNA into a single stranded one 95 degrees
PCR elongation
Building up a new DNA strand using DNA polymerase 72 degrees
Gel electrophoresis
A technique that is used to separate different molecules. It used to analyse proteins and DNA. This technique involves placing the molecules into wells cut into agarose gel and then applying an electric field, they will then separate
Factors that effect movement in gel electrophoresis
Net charge - negatively charged molecules move towards the anode and positively charged molecules move towards the cathode. Highly charged molecules move faster.
Size - smaller molecules move faster
Composition of gel - size of the pores determine the speed they move
Electrophoresis of proteins
Carried out at a constant pH using a buffer solution, as the amino acid can become ever positive or negatively charged. Is used to separate the polypetides produced by different alleles of many genes. For example, it can be used to test if someone has sickle cell anaemia as they will do the test on their beta globin, as this i different in people who have sickle cell anaemia
Electrophoresis of DNA
DNA fragments have a small charge due to the phosphate group, these fragments move torwards the anode. They choose a piece of DNA which varys between different people, these often contain variable numbers of repeated DNA sequences and are known as VNTRs. PCR can be used to increase DNA quantity. Two different restriction enzymes cut the DNA, they are then put into gel and separated by an electric field. To make the fragments visible, they are transferred onto absorbant paper, a radioactive probe is added to bind to the invisible bands of DNA, so that they blacken an X-ray film
Production of insulin
The mRNA with the code for human insulin is identified and isolated. This was then incubated with enzyme reverse transcriptase, this enzyme used reverse transcription to make single stranded cDNA. This was converted to a double stranded DNA molecule using DNA polymerase to form a complimentary strand. This DNA was with the same restriction enzyme which was used to cut the plasmid from the bacteria, to produce complimentary sticky ends. The insulin DNA and plasmid DNA are mixed together with a ligase enzyme. The sticky end bases form hydrogen bonds. A recombinant plasmid is formed. The recombinant plasmid is introduced to the bacteria, a transformed bacterium is identified. The gene is cloned by growing the bacterium. The transformed bacteria can now produce insulin. These bacteria are now grown on an industrial scale in large fermenters. Insulin is then extracted and purified.
Restriction endonucleases
A class of enzymes called restriction enzymes which cut at specific target sites on DNA . They break the sugar-phosphate backbone to create sticky ends, which are lengths of unpaired bases. They can the form hydrogen bonds with DNA cut from the same restriction enzyme. Used to cut and isolate segments of DNA
Reverse transcriptase
mRNA is collected and incubated with the enzyme reverse transcriptase which is used to convert mRNA to cDNA. These are then converted to double stranded DNA molecules using DNA polymerase to create a complimentary strand.
Properties of plasmids that allow then to be used in genetic technology
A low molecular mass so they are readily taken up by bacteria. An origin of replication so that they can be copied. Several single target sites for different restriction enzymes in a short length of DNA called a polylinker. One or more marker genes, allowing identification of cells that have taken up the plasmid.
DNA ligase
When the opened plasmid and length of DNA are mixed together, some of the plasmids sticky ends pair up with the sticky ends on the new gene. The enzyme DNA ligase is used to link the sugar phosphate backbone of the DNA molecule and the plasmid.
Microarray
Used to identify the genes present in an organisms genome and to find out which genes are expressed within a cell.
Cystic fibrosis
Cystic fibrosis is caused by a mutation on the CFTR gene, which makes the CFTR protein defective. Genetic technology can be used as a treatment for cystic fibrosis by inserting the normal dominant allele of the CFTR gene into the DNA of cells in the respiratory system. The gene can be inserted into a vector such as liposomes or harmless viruses and then taken as a spray. However, not all cells took up the virus and there were some unpleasant side effect, such as the patients being infected by the virus. The effects of the liposomes were also short lived and needed repeating
