3.8.4 Gene Technologies Flashcards
What’s recombinant DNA technology
The transfer of fragments of DNA from one organism/species to another
Why does transferring DNA fragments from one organism to another allow for the DNA to be expressed
Genetic code is universal (the same DNA triplets code for the same amino acids in all organisms), so are the transcription and translation mechanisms
This allows the transferred DNA to be be transcribed and translated within cells of the recipient organism
What are the stages in producing proteins using DNA technology and cloning
- Isolation of DNA fragments containing the gene for the desired protein
- Insertion of the DNA fragment into a vector
- Transformation, transferring DNA into a host cell
- Identification of host cells which have successfully taken up the gene by using gene markers
- Cloning the population of host cells
Three methods for producing DNA fragments
Conversion of mRNA to complimentary DNA (cDNA) using reverse transcriptase
Using restriction enzymes to cut a fragment of the desired gene from DNA
Creating the gene in a gene machine
How is a DNA fragment produced using reverse transcriptase
A cell which readily produce the protein is selected eg beta cells in the pancreas for insulin
The cells have large quantities of relevant mRNA which is easily extracted
Reverse transcriptase is used to make DNA from RNA, this is cDNA as it has nucleotides complimentary to mRNA
Enzyme DNA polymerase is used to build complimentary nucleotides to cDNA
Produces doubles stranded DNA with required gene
What’s restriction endonuclease
An enzyme which bacteria produce which cut up viral DNA if injected into the bacteria cell
How is a DNA fragment produced by restriction endonuclease
Cuts DNA at a specific palindromic bas sequence with a staggered cut leaving sticky ends
How are DNA fragments produced from a gene machine
nucleotide base sequence is determined from desired protein wanting to be produced, amino acid sequence is known, mRNA codons are looked up and complimentary DNA triplets are worked out (working backwards)
This sequence is fed into a computer, oligonucleotides assembled into a gene
This gene doesn’t contain introns
The gene is replicated using a polymerase chain reaction forming a double stranded gene
What’s in vitro
Using the polymerase chain reaction
What’s in vivo
Transferring DNA fragments to a host cell using a vector
What’s an in vitro method for amplifying DNA fragments
Uses PCR (polymerase chain reaction)
DNA strand is seperated, temp at 95°c, hydrogen bonds break
Annealing, 55°, primers anneal complimentary bases at ends of DNA fragment, provide start sequence for DNA polymerase and prevent two separate strands from rejoining
Synthesis of DNA, 72° builds complimentary strands of DNA, 2 copies of OG fragment
in vivo to amplify DNA fragments
Extra lengths of DNA are added to the DNA fragment
RNA polymerase and transcription factors attach to the promoter region
Terminator added to other end of DNA, releases RNA polymerase to stop transcription
Restriction endonuclease is used to break the plasmid loop (plasmid acts as a vector), this needs to be the same enzyme that cuts out the DNA fragment to ensure the sticky ends on the plasmid are the same as the sticky ends on the DNA fragment
DNA fragment is incorporated into the plasmid, DNA ligase forms phosphodiester bonds, plasmid has recombinant DNA
How are host cells transformed after in vivo
Transformation, plasmids reintroduced to bacteria by mixing into a medium of Ca 2+, membrane more permeable allowing plasmid to move into cytoplasm
Not all bacteria take up the plasmid as the close up or the DNA fragments join forming their own plasmid
What are marker genes
Identify whether a gene has been taken up by bacterial cells
How are marker genes used to identify GM cells
The DNA fragment is placed in between a gene for antibiotic resistance, this gene stops working
Replica plating grows bacteria with the antibiotic they are no longer resistant too, this kills the colonies on the replica plate however we can identify them and clone the transformed cells from the master plate
What are benefits and issues with recombinant DNA technologies for agriculture industry and medicine
Agriculture
GM crops can survive extreme weather
GM crops can prevent deficiencies like vitamin A in golden rice
Consequences of genetic engineering can’t be predicted
Industry
Microorganisms can control pollution from factories
Is money better spent on other issues like hunger than on DNA technologies
Medicine
Microorganisms produce hormones to cure disease
Produce drugs cheaply
Antibiotics have added resistant genes, they could spread to harmful bacteria
Similarities between DNA technology and gene therapy
Hhh
What’s a DNA probe
Short single stranded length of DNA with a radioactive or fluorescent label making it easily identifiable
How are radioactive and fluorescent probes identified
Radioactive probes identified using X-ray film exposed by radioactivity
Fluorescent probes emit light once it binds to the target DNA sequence
What is DNA hybridisation
Section of DNA or RNA is heated until it becomes single stranded, combined with a single stranded section of DNA with a complimentary base sequence these two complimentary strands anneal reforming a double strand
How are specific alleles of a gene identified using DNA probes and hybridisation
Determine the base sequence of the mutant allele using DNA sequencing techniques
A DNA fragment with a complimentary base sequence to the mutant allele is produced
Copies of the DNA probe are made using the polymerase chain reaction
The DNA probe has a marker such as radioactive isotopes or fluorescent dye
DNA from the person being tested is located and heated separating it into 2 strands, these strands are cooled in a mixture containing DNA probes
If the mutant allele is present the DNA probe binds to it due to the complimentary base sequence
DNA is washed to remove unattached probes
Hybridised DNA is fluorescently/radioactively labelled, a light or autoradiography is used to detect the mutant allele using
What else can DNA probes be used for
Screen patients for heritable conditions
Drug responses
Health risks
What is genetic counselling and how does it use info from genetic screening
Counsellors give advice and info enabling people to make decisions for themselves or offspring
Screenings detect types of disease and gene changes to whether a patient will benefit from a treatment, provides a basis for the discussion
What’s personalised medicine
Doctors provide advice and care based on an individuals genotype
Genes can determine if a drug is more or less effective, doctors can prescribe better dosages saving money
