manipulating genomes and genetic engineering Flashcards
what is a DNA probe
a small complimentary section of code of a gene you are looking for. contains fluorescent marker or radioactive dye.
what are primers
short pieces of single stranded DNA that are complimentary to the bases at the start of the DNA fragment you want.
outline the 3 stages of PCR
- heat to 95 degrees, breaks hydrogen bonds between 2 strands of DNA
- cool to 55 degrees, allows primers to anneal
- heat back to 72 degrees, free complimentary nucleotides are joined by taq polymerase which forms sugar phosphate backbone
what are the 2 methods scientists can use to isolate a gene
restriction endonucleases, - restriction enzymes that cut at specific sites isolating the gene and leaving sticky ends. different restriction enzymes cut at different specific sites.
reverse transcriptase which converts MRNA of complimentary gene to cDNA
outline the steps of setting up gel electrophoresis
gel tray placed into gel box. wells are at negative (cathode) end. buffer solution is added to reservoirs to ensure solution carries current + maintain pH.
DNA sample is loaded into each well using a micropipette and loading dye is added to ensure the sample sinks making it easier to see.
place lid on box and connect to power supply, turn on and leave electrical current to pass through gel
what is the gel called in gel electrophoresis
agarose gel
which way will DNA fragments move in gel electrophoresis and why
will move from the cathode to the anode as DNA fragments are negatively charged (phosphate group) and so travel to positive end.
how are DNA fragments separated in gel electrophoresis
by size, smaller fragments travel further.
what is done with proteins before gel electrophoresis is used and why
denatured with a chemical so that they all have the same charge
what does southern blotting involve
nylon sheet is placed on top of gel trat, DNA fragments are soaked onto nylon sheet
what is hybridisation
fluorescent dye is added to fragments and they are viewed under UV light
or
radioactively labelled dye is added to fragments and viewed under X-ray.
what is a genome
all the genetic material in an organism
which is smaller microsatellites or minisatellites
microsatellites
what is unique in each persons genome that allows DNA profiling to occur
satellite pattern - these are non coding sequences that repeat at a specific loci, the number of repeats is unique to each individual so each person has a different satellite pattern.
what are the different uses of DNA profiling
used in forensic analysis to compare crime scene evidence to potential suspects
used in paternal cases
used in medical diagnosis - analyses the risk of genetic disorders
eg. compares DNA profile of embryo to parents DNA profile, if the profiles match the embryo has inherited the disorder. - used to screen for cystic fibrosis.
what are transformed organisms
organisms that have recombinant DNA - DNA formed by joining together DNA from different sources.
what is a transgenic organism
an organism that has been genetically engineered to include a gene from a different species
what is a plasmid
small circular molecule of DNA in bacteria
what is a bacteriophage
virus that can infect bacteria
outline the genetic engineering process
- isolate desired gene/DNA fragment using restriction enzymes
- vector DNA (plasmid) is cut upon using the same restriction enzymes (so sticky ends are complimentary)
-vector DNA and DNA fragment is mixed together with DNA ligase - forms sugar phosphate backbone
-the vector is used to transfer the gene into bacterial cells, host cells are persuaded to take in the plasmid by electroporation/heat shock/electrofusion
how does a bacteriophage vector transfer the gene to the host
infects the host bacterium by injecting its DNA into it, the phage DNA then integrates into the bacterial DNA
what happens during heat shock
plasmid and bacteria are placed into solution containing Ca2+ ions, temperature is increased. this increases permeability of bacterial membrane + kinetic energy
in genetic engineering of plants, what is the name of the bacterium in which the plasmid is taken from
Argobacterium tumefaciens
list 3 advantages of genetically modified crops
- reduces amount of pesticides farmers have to use which can harm environment (if insect resistent modified)
- GM plants can be designed to be more nutritious
- increases the crop yield
list 3 disadvantages of genetically modified crops
- may encourage monoculture - decreases biodiversity
- if all genetically identical whole crop is susceptible to disease
- could interbreed with wild plants creating ‘super weeds’
what is name of the term given to producing drugs from animals
pharming
outline how the protein antithrombin can be produced by goats
- DNA fragments that code for production of protein in mammary gland are extracted
- dna fragments are injected into a goat embryo
- embry implanted into surrogate mother
4.if offspring produces the protein, selective breeding is sused to produce a herd of goats that produce the protein in their milk. which can be extracted
what are 2 benefits of pharming
- drugs can be made in large quantities
- this makes them more available to people
what are 2 disadvantages of pharming
concern manipulating animal genes could have. harmful side effects for animal
enforces idea that animals are merely ‘assets’ we can use.
how have pathogens been used in research
tumour cells have receptors on membranes for poliovirus meaning the virus recognises and attacks tumour cells.
scientists have. manipulated the poliovirus to inactivate the genes that cause the disease, so can use poliovirus to attack and kill cancer cells without infecting the person
what is an advantage of using pathogens in research
previously untreatable diseases can now be treated, reduces the suffering. they cause
what is a disadvantage of using pathogens in research
some people are worried it could cause an outbreak
The GM version could revert back to origional form and cause outbreak
in wrong hands could be used maliciously to create agents for biowarfare.
what are the 2 types of gene therapy
somatic
germ line
how would a genetic disease caused by 2 recessive alleles be treated
a working dominant allele could be added. eg by using vectors
how would a genetic disease caused by a dominant allele be treated
by silencing it - sticking a bit of DNA in middle of allele so it doesn’t work anymore.
why is a virus the most common vector used in genetic engineering
it has the mechanism to recognise cells and deliver genetic material into them.
what is somatic therapy
inserting functional alleles into body cells, can be:
ex-vivo: new gene is inserted via a virus vector into cells outside the body. eg. bone marrow cells are extracted and exposed to virus which inserts gene into cells. these cells are grown in a lab and returned via injection
in vivo: new gene is inserted via a vector into cells inside the body.
is somatic therapy long term, can offspring inherit the genetic disorder
somatic therapy is not long term as only alters body cells which die and become replaced, therefore patients need to return for multiple treatments.
somatic therapy doesn’t affect the individual sex cells so any offspring can still inherit the disorder
what is germ line therapy
inserting functional alleles into zygotes or gametes - every offspring produced will be alered by the gene therapy.
what is DNA sequencing
determining the sequence of nucleotides in a piece of DNA
what are terminator bases
bases that have a hydrogen atom instead of OH group on 3’ end. this means they cannot form phosphodiester bonds with the next nucleotide .
outline the steps of pyrosequencing
- section of DNA is cut into fragments, a strand from each fragment is attached to a small bead
- PCR amplifies the DNA fragments on each bead
- each bead is placed into a separate well
- free nucleotides are added
- wells contain specific enzymes which cause light to be emitted when a nucleotide is added
- computers analyse the occurrence and intensity of light and process to interpret DNA sequence
