A2 Genome Projects and Gene Technologies Flashcards
What is recombinant DNA technology?
Transferring DNA fragments from one organism to another
Describe the use of reverse transcriptase in making DNA fragments
- mRNA molecules used as template
- RT makes DNA from RNA template, producing cDNA
- mRNA isolated from cells, mixed with free DNA nucleotides and RT
- RT uses mRNA as template to synthesis new strand of cDNA
Describe the use of restriction endonuclease enzymes in making DNA fragments
- Palindromic sequences of nucleotides
- RE recognise specific palindromic sequences (recognition sites) and cut (digest) DNA at these places
- RE’s are specific to RS - shape of RS is complementary to RE’s active site
- If RS’s present either side of fragment, use RE to separate from DNA
- DNA sample incubated with specific RE, cuts DNA fragment out via hydrolysis reaction
- Cut can leave sticky ends - unpaired bases each end of fragment - can be used to bind fragment to another bit of DNA with sticky ends with complementary sequences
Describe the use of a gene machine in making DNA fragments
- Sequence required is designed
- 1st nucleotide fixed to support (bead)
- Nucleotides added in correct order, including adding protecting groups - make sure they are joined at right places, prevent unwanted branching
- Oligonucleotides - short sections of DNA produced. Broken from support and protecting groups removed. Then joined together to make longer fragment
In vivo amplification - describe the process of how DNA fragments are inserted into a vector
A vector used to transfer DNA into cell. Can be plasmids or bacteriophages (viruses that infect bacteria)
- Vector DNA cut open using same RE used to isolate fragment with target gene. So sticky ends of vector are complementary to sticky of fragment
- Vector DNA and DNA fragment mixed together with DNA ligase - joins sticky ends together - called ligation
- New combination of bases in DNA called recombinant DNA
In vivo amplification - describe how the DNA fragment is transferred into a host cell
- Plasmid: host cells persuaded to take in plasmid - e.g use of temp. to make walls of host more permeable
- Bacteriophage: infects host bacterium by injecting its DNA into it - phage DNA integrates into bacterial DNA
- Host cells that take up vectors are now transformed
In vivo amplification - describe how you would identify transformed host cells
- Marker genes inserted into vector at same time as gene - any transformed host cells contain gene and marker gene
- Host cells grown on agar plates - divide and replicate DNA - creates colony of cloned cells.
- Marker gene can code for antibiotic resistance - host cells on agar contain antibiotic - only transformed cells with marker gene survive and reproduce.
Marker gene can code for fluorescence - agar placed under UV light, transformed cells with fluoresce - Identified transformed cells allowed to grow, producing lots of copies of cloned gene
What are promoter regions and how are they used in amplifying DNA fragments?
Short DNA sequences that tell RNA polymerase to start producing mRNA. They are complementary to bases at the start of the DNA fragment
What are terminator regions and how are they used in amplifying DNA fragments?
Short DNA sequences that tell RNA polymerase when to stop transcribing mRNA
Describe the process of in vitro amplification using the PCR
- Mixture containing DNA sample, free nucleotides, primers and DNA polymerase
- Mixture heated to 95 degrees C- break hydrogen bonds between DNA strands
- Mixture cooled to around 55 degrees C - primers can anneal to strands
- Mixture heated to 72 degrees C - taq polymerase can work
- Taq polymerase lines up free DNA nucleotides alongside template strands - specific base pairing - new complementary strands formed
- Two new copies of fragment formed - one cycle of PCR complete
- Each PCR doubles amount of DNA
How can recombinant DNA technology be used to benefit humans in agriculture, industry and medicine
Agriculture: crops transformed to give higher yields and more nutrition - reduce risk of famine and malnutrition. Also to have pest resistance - less pesticides needed - reduces costs and environmental problems (leaching)
Industry: enzymes produced - large quantities for less money - reduces costs
Medicine: drugs and vaccines produced - made quickly, cheaply and in larger quantities
What are the concerns with the use of recombinant DNA technology in agriculture, industry and medicine?
Agriculture:
- Monocultures of transformed plants may be used - whole crop vulnerable to same disease - genetically identical. Monocultures reduce biodiversity
- ‘Superweeds’ - weeds resistant to herbicides. Could occur if transformed crops interbreed with wild plants - uncontrolled spread of recombinant DNA
- Organic crops could be contaminated by wind-blown seeds of genetically modified crops - can’t sell as organic
Industry:
- Few large biotechnology companies control forms of genetic engineering - use increases, may force smaller companies out of business
- Without proper labelling - may not have choice whether to consume food from genetically engineered organisms
Medicine:
- Companies owning technologies may limit use that could save lives
- Technology could be used unethically - designer babies
What is the humanitarian view of recombinant DNA technology?
- Crops produced could reduce risk of famine and malnutrition - drought-resistant crops
- Crops used to produce useful pharmaceutical products - drugs more available to people
- Medicines produced more cheaply - more affordable
What is gene therapy and how is it used?
Involves altering defective genes (mutated allele) inside cells - treat genetic disorders and cancer
If disorder caused by:
- Two mutated recessive alleles - add working dominant allele
- Mutated dominant allele - ‘silence’ allele (e.g. sticking bit of DNA in middle of allele so it doesn’t work)
Both processes involve insertion of DNA fragment into original DNA
In gene therapy, how is a ‘new’ allele (DNA) inserted inside cell?
- Allele inserted into cells using vectors (same as recombinant DNA technology)
- Different vectors used - altered viruses, plasmids or liposomes
