MG (Genetic engineering) Flashcards
what’s DNA ligase
enzyme that catalyses the joining of sugar and phosphate groups (phosphodiester) within DNA via condensation reaction
DNA polymerase vs DNA ligase
DNA polymerase adds nucleotides to the template strand (5’ to 3’), and ligase seals the gaps between the nucleotides on lagging strand (3’ to 5’) by joining Okazaki fragments
what are plasmids
small loops of DNA in prokaryotic cells
what is Recombinant DNA
a composite DNA molecule created in vitro by joining foreign DNA with a vector molecule (plasmid)
What’s genetic engineering
when genes are isolated from one organism and inserted into another organism, using vectors.
What are the main steps in genetic engineering
- The required gene is obtained
- A copy of the gene is placed inside a vector
- The vector carries the gene into a recipient cell
- The recipient expresses the novel gene
Step 1 - How is the required gene obtained in genetic engineering
- mRNA obtained from cells where the required gene is being expressed. Reverse transcriptase catalyses formation of a single strand of complementary DNA (cDNA) using the mRNA as a template. The addition of primers and DNA polymerase can make this cDNA into a double-stranded length of DNA, whose base sequence codes for the original protein.
- If scientists know the nucleotide sequence of the gene, then the gene can be synthesised using an automated polynucleotide synthesiser.
- If scientists know the sequence of the gene, they can design PCR primers to amplify the gene from the genomic DNA.
- A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes.
what’s a vector
in gene technology anything that can carry/insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria.
Step 2 - How is the gene placed into a vector
- Plasmids mixed with restriction enzymes that will cut the plasmid at specific recognition sites.
- The cut plasmid has exposed unpaired nucleotide bases, called sticky ends
- If free nucleotide bases, complementary to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal. DNA ligase enzyme catalyses the annealing.
- A gene may be sealed into an attenuated (weakened) virus that could carry it into a host cell.
What are the 5 methods used to insert vector into the recipient cell
- Heat shock treatment
- Electroporation
- Electrofusion
- Transfection
- T1 (recombinant) plasmids
What’s electrofusion
electrical fields help to introduce DNA into cells.
What’s electroporation
a pulse of electricity makes the recipient cell membrane more porous.
what’s heat shock treatment ?
- bac subjected to alternating periods of cold and hot in the presence of calcium chloride,
- their walls and membranes will become more porous and allow in the recombinant vector.
- Ca2+ reduces repulsion between -vely charged parts of DNA and phospholipids
what’s transfection
DNA can be packaged into a bacteriophage, which can then transfect the host cell.
How are T1 (recombinant) plasmids used to get vector into recipient cell
the plasmids are inserted into the bacterium Agrobacterium tumefaciens, which infects some plants and naturally inserts its genome into the host cell genomes.
How is reverse transcriptase used
- Find a cell that produces the protein you require (e.g β cells of the Islets of Langerhans in the pancreas produce insulin).
- Extract the mRNA from the cells.
- Use reverse transcriptase to make cDNA from the RNA.
what’s the direct method of introducing gene into recipient
- If plants are not susceptible to A. tumefaciens, then direct methods can be used.
- Small pieces of gold or tungsten are coated with the DNA and shot into the plant cells. This is called a “gene gun”.
What’s genetic engineering also known as?
- recombinant DNA technology
- genetic modification
function of reverse transcriptase found in retroviruses (HIV)
- HIV contains RNA that they can inject into the hosts genome
- reverse transcriptase catalyses formation of cDNA using RNA as template
- useful in genetic engineering
restriction enzymes create ……… and ……….. ends
- sticky (staggered cuts)
- blunt (cuts not staggered)
How is insulin obtained from GM bacteria
- adding reverse transcriptase enzyme makes a single strand of cDNA and treatment with DNA polymerase makes a double strand - the gene
- addition of free unpaired nucleotides at the ends of DNA produces sticky ends
- with the help of ligase enzyme, the insulin gene can be inserted into plasmids extracted from E.coli bac ( now recombinant DNA)
- Bac mixed with recombinant plasmids and subjected to heat shock in presence of CaCl2, so they’ll take up the plasmids
what are the two antibiotic resistance genes in bacterial plasmids
- resistance to tetra
- resistance to amp
How do you find the plasmids that have taken up the desired gene?
- desired gene is inserted into the plasmid midway through the gene for tetra resistance.
- Some plasmids take up the gene some do not
- all colonies grow in amp plate (as all plasmids have gene for amp resistance)
- Bacteria only grow on the tetracycline plate if they have NOT got the insulin gene.
How is the risk of transgenic bacteria escaping into the wild controlled?
- genes knocked out so they can’t synthesise particular nutrients so will only survive in the lab where they are supplied those nutrients in growth medium
Benefits of GM microorganisms (E.coli)
- insulin production for treatment of diabetes
- make human growth hormone to treat children with pituitary dwarfism
Hazards of GM microorganisms (E.coli)
- can escape into wild + transfer marker genes for antibiotic resistance to other bac
- HOWEVER, bac modified so can’t grow outside of lab
Benefits of GM Bt tobacco and Bt maize
- genetically modified to produce toxin to kill pests do don’t need to spray it, other organisms not contaminated
Hazards of GM Bt tobacco and Bt maize
- Bt is toxic to monarch butterflies
- HOWEVER, they don’t take nectar from maize or tobacco plants in the wild
Benefits of GM soya beans
genetically modified to be resistant to herbicide
Hazards of GM soya beans
- superweeds may arise
- HOWEVER, this hasn’t happened
Benefits of GM golden rice
rice modified to contain beta carotene (precursor to vitamin A ), preventing blindness
Hazards of GM golden rice
- concerns about farmers having to buy seeds every year
- HOWEVER, companies offered free licenses
Benefits of GM plantain
- enhanced to contain more zinc to combat zinc deficiency
- zinc is an enzyme cofactor
Hazards of GM plantain
- inserted genes will be expressed in us
- HOWEVER, all the food we eat contains genes + we digest them
Benefits of GM crop plants resistant to pests
- no need to use pesticides
Hazards of GM crop plants resistant to pests
- people die due to exposure when applying them
- concerns that farmers wouldn’t want GM seeds and wouldn’t have the choice to buy non-GM seeds.
- HOWEVER, non-GM seeds are stills sold
Benefits of GM pathogens
- used as vectors in gene therapy
- modified to have no virulence so can be used in vaccines without making patient ill
Hazards of GM pathogens
- in gene therapy, allele may be inserted into the genome in a way that increase risk of cancer or interfere with gene regulation
Benefits of GM mice
- used to develop therapies for breast and prostate cancer
- certain genes knocked out in them so their function can be determined
Hazards of GM mice
- some object to the use of animals for testing
- HOWEVER, there are strict regulations that govern their welfare + we have benefited from usage of animals
what’s ‘pharming’
- Creating animal models.
- Adding/removing genes so that the animal develops certain diseases to act as models in the development of new treatments
- Eg. ‘Knock out mice’ have genes deleted so that they develop cancer. Treatments can then be trailed. - Creating human proteins.
- Human gene can be inserted into a fertilised egg (cow/pig/sheep).
- Promotor sequence could mean that the gene is only expressed in the mammary glands – milk will then contain the required human protein.
What are palindromic sequences?
sections of DNA that consist of ANTIPARALLEL BASE PAIRS (base pairs that read in the opposite direction).
Explain cutting out DNA fragments using restriction enzymes
- Restriction enzymes recognise specific palindromic sequences (known as recognition sequences) and cut the DNA at these places. Different restriction enzymes cut at different specific palindromic sequences, because the shape of the recognition sequence is complementary to an enzyme’s active site.
- If the recognition sites are present at either side of the DNA fragment you want, you can use restriction enzymes to separate it from the rest of the DNA.
what’s the role of Restriction endonucleases
cut DNA at certain recognition points via hydrolysis