Gene technology Flashcards
what is recombinant DNA technology
involves inserting a foreign gene into an organism’s genome, resulting in the expression of the new gene.
Describe some applications of gene technology in medicine, agriculture and industry.
- Use of gene products.- GM microbes produce a protein for medical, agricultural or industrial use
- Alteration of phenotype.- An organism’s characteristics can be altered – mostly used in crops or the production of animals
- Gene therapy.- Using gene technology to treat a disease
what are the steps to forming recombinant DNA
- restriction enzymes cut dna at specific recognition sites to create either sticky or blunt ends to seperate a certain gene
- cut the bacterial genome with the same restriction enzyme so that its complimentary
- the target gene and the bacterial genome have complimentary sticky ends
- bases join by weak hydrogen bonds and sugar phosphate backbone formed by dna ligase
- forms recombinant dna
what is gel electophoresis
separates fragments after a gene has been cut by restriction enzyme
describe the steps to gel electrophoresis
- tank has postive and negative elctrode at each end
- DNA has a n overall negative charge due to phosphate so its pulled to positive
- smallest travels furthest
define PCR
a technique that is used to amplify one sample of DNA thousands of times over to create a large enough DNA sample for extensive analysis using a thermocycler
It is an In Vitro amplification of DNA.
what are the steps to PCR
- heat to 95- breaks the weak H bonds between bases so strands seperate (DNA denatured)
- Mixture cooled to 55, primers, free nucleotides Taq polymerase added
- primers anneal with their complimentary bases- DNA polymerase attaches
- raise temp to 72- optimum for polymerase- binds nucleotides
- repeat- DNA doubles each time
what are some uses of PCR
- Recombinant DNA technology,- RT-PCR (Reverse transcriptase PCR)
- Detecting genetic diseases
- Genetic fingerprinting
what is dna sequencing
a lab technique that determines the order of chemical bases in DNA molecule
describe the steps to manual sanger sequencing
- four seprate PCR reactions happen, one for each ddNTP (ddATP, ddCTP, ddGTP, ddTTP)
- DNA strands from each PCR are run in 4 seperate lanes of gel
- user reads all 4 lanes of gel at once , from bottom to top,
- lanes are used to determine the terminal ddNTP for each band
Describe the steps to automated sanger sequencing
- Each ddNTP is fluorescently labelled as either, ddATP, ddTTP, ddGTP, and ddCTP
- all ddNTPs are combined into one PCR reaction
- all oligonucleotides are run in single capillary gel electrophoresis within the sequencing machine
- computer reads each band of the capillary gel, in order, using fluorescence to identify of each terminal ddNTP
what is DNA profiling/fingerprinting
- Introns contain minisatellites- repeating sequences of genes.
- minisatellites are regions of hypervariabilty (VNTRs)
- minisatellites vary in number of repeats but people who are related are more likey to have the same number of repeats
- can be used to determine paternity.
- crime scenes to identify DNA
define DNA probe
short strand of DNA, base sequence complimentary to target gene, radioactive/ fluorescent labelling
what are the steps to gentic profiling/fingerprinting
- collection and extraction
- buccal swab- break cells
- proteins removed and DNA extracted using ice cold ethanol - Digestion
- restriction enzymes cut DNA into bits - enzymes shouldnt cut at minisatellites. - Seperation
- seperated by length via gel electrophoresis
- the DNA is made single stranded by immersing into alkali solution
- place nylon sheet on top of gel the place blotting sheet on top of nylon- DNA is now on nylon. - Hybridization
- probe is added, binds to complimentary sequence wherever its encountered
- probe contaions radioavtive label
- take an autoradiograph - development
- X ray image
define gene cloning
Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism (sometimes called sub-cloning, because only part of the organism’s DNA is being cloned.)
what are the overall steps of gene cloning
- isolation - of DNA containing the target gene
- insertion - of DNA into vector
- transformation - transfer of DNA into a suitable host
- identification - finding those host organisms containing the vector and DNA using gene markers
- cloning - of the successful host cells
Describe the isolation step using restriction enzymes
- restriction enzymes cut genes at specific recognition sites to isolate the target gene
- recognition sites are normally palindromic
- forms sticky ends which are complimentary to each other
- They will join with another sticky end (i.e. from a gene to be inserted) but only if it has been cut with the same restriction enzyme.
- Sticky ends are joined together using DNA ligase
- forms recombinant dna
describe isolation step in gene cloning using reverse transcriptase
-
Modification: Eukaryotic DNA is first transcribed into mRNA
- The mRNA is then modified as necessary and then obtained from the eukaryotic cell e.g once introns removed
- RNA however cannot be inserted and used for transcription- a dsDNA is need for this - Reverse transcriptase: RNA → complimentary DNA (cDNA)
- DNA polymerase : cDNA → dsDNA
describe the insertion step of gene cloning
- the plasmid is cut by the same restriction enzyme as DNA
- forms complimentary sticky ends
- The target gene can be inserted and joined by dna ligase
- Genetic transformation occurs when a cell takes up DNA and expresses the genes on that DNA. Many different types of cells can be transformed – plant, animal, human, bacterial.
- bacteria divides and new bacteria gets plasmid
- genes are transcribed and translated to make protein
what are vectors
things that carry DNA into host
Describe plamids as vectors
- Used to carry desired DNA into host cell
- Small, circular dsDNA
- Plasmid vectors can be designed with a variety of features:
-Antibiotic resistance
-Colorimetric “markers”
-Strong or weak promoters for driving expression of a protein
define transgenic organism
an organism that has had its DNA altered by introducing foreign DNA from another species.
define transformation
The process of introducing the foreign DNA into a prokaryotic host cell
describe the transformation step in gene cloning - heat shock
- cells and vectors incubated together
- dna is negatively charged
- CaCl2 transformation solution
- Ca shields negative charge
- incubate on ice for 10 mins
- heat shock at 42 degreees for 50 secs
- incubate on ice for 2 mins
- some cells take up vectors
describe the transformation step in gene cloning - electroporation
- cells and plasmids are mixed together
- an electrical pulse is applied, causing pores to form
- plasmids eneter the cell through the pores
- pores seal with the plasmid insode transformed cell
describe how the identification step happens in gene cloning
Gene markers can be:
- Resistance to an antibiotic
- A fluorescent protein
- An enzyme whose action can be identified
Usually a gene marker is disrupted if the DNA fragment is present.
how do antibiotic resistant gene markers work
- Often plasmids used often carry two genes to provide the bacteria resistance to ampicillin & tetracycline.
- if it is resistant to ampicillin it contains the plasmid, if it is not resistant to tetracycline, it contains the recombinant plasmid as the placement makes the antibiotic resistant gene non function
what can act as vectors
plasmids, viruses, liposomes, stem cells
what are some uses of cloning
- Research – cloning of genes to study gene function (a lot done in mice)
- Improvement of crop yields
- Production of human proteins
- Production of animals with useful traits
- Treatment of genetic defects through gene therapy
what is gene therapy
“the introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders”
what are probelms with gene therapy
- Identifying the problem.
- Getting DNA into the cell.
- Targeting right cells.
- Getting genes to right part of cell.
- Stability.
- Activating the gene
- Avoiding harmful side-effects
how is somatic cell nuclear transfer different to gene therapy
- Gene therapy involving somatic cells may be therapeutic, but the genetic changes produced are not inherited.
- transfection of stem cells, such as in the bone marrow achieves longer persistence of therapy in patients.
- The introduction of altered genes into the germline (reproductive) cells enables changes to be inherited
how can human insulin be mass produced
- Identify and synthesise the human gene
- Isolate plasmids from bacteria
- Insert synthetic DNA into plasmids
- Insert plasmid into bacterial cell
- Identification of bacteria with the plasmid
- Make the functional protein
what are the advantages of transgenic organisms
- Fewer side effects
- Reduced incidence of rejection
- Reduced risk of disease
- Cheaper
- No ethical issues – Insulin used to be extracted from the pancreas of cows, pigs and dogs.
what are some disadvantages of transgenic organisms
- There was some evidence of reduced hypoglycaemic awareness in some patients switching from porcine/bovine insulin to human, which in some cases lead to death.
- Technical difficulty with procedure, for example uptake of plasmids by bacteria (about 1%), controlling the expression of the recombinant gene effectively and ensuring the protein folds correctly
- Infection risk – Making sure that the final product is entirely free of the host cells used
- Sterility also paramount
- Avoiding use of animal products in culture medium
- There is increasing use of insect and mammalian cells to prevent bacterial contamination
- Public perception of GM products
