Recombinant DNA Technology Flashcards
Suggest why it is possible to transfer DNA from one organism to another
- genetic code is universal
- transcription/ translation mechanisms are universal
Transgenic
genetically modified organism
State methods of gene isolation
- restriction endonucleases
- reverse transcriptase
- gene machine
Describe how genes are isolated using restriction endonucleases
- active site binds to complementary palindromic recognition site
- cuts by hydrolysis of phosphodiester bond
- staggered cut produces sticky ends
Describe how genes are isolated using reverse transcriptase
- reverse transcriptase binds to primer on mRNA
- transcription occurs with mRNA acting as template to form cDNA
- DNA polymerase copies cDNA strand forming double-stranded cDNA
Describe how a gene machine is used to artificially synthesise genes
- computer designs oligonucleotides (short strands of nucleotides) using known base sequence
- DNA polymerase joins oligonucleotides to form desired gene
Evaluate use of reverse transcriptase to isolate genes
- mRNA abundant in cytoplasm
- no introns so can be transcribed by prokaryotic cells
- involves multiple steps so time consuming
- mRNA technically difficult to extract from cytoplasm
- no regulatory genes
Evaluate use of restriction endonuclease to isolate genes
- create sticky ends to easy to insert into plasmid
- isolated DNA contains introns so cannot be transcribed by prokaryotic cells
- difficult to find gene of interest since exact locus must be known
Evaluate use of gene machine to isolate genes
- ability to create any gene
- no introns so can be transcribed by prokaryotic cells
- requires specialist equipment
- must know exact sequence
Sticky End
- formed by restriction enzymes at a palindromic sequence
- exposed nucleotides at end of DNA fragment
- complementary to each other
Describe how to prepare a culture of transformed host cells as an in vivo method to amplify DNA fragments
- cut desired gene from cell using restriction endonuclease
- cut plasmid with same restriction endonuclease
- (add promoter and terminator regions to fragments of DNA)
- use DNA ligase to join
- sticky ends attach
- use Ca2+ and heat shock
- plasmid enters bacterial cells
Describe the process of transformation in gene transfer
- medium containing Ca2+
- heat/electric shock
- membrane permeable so plasmids enter
Suggest why bacteria cells do not always take up desired gene
- not all plasmids enter bacterial cells
- some plasmids close up without incorporating DNA
- some DNA fragments join up to form plasmids
Maker Genes with examples
genes used to identify cells that have taken up desired gene, e.g. antibiotic resistance, fluorescent, enzyme
Replica Plating
technique to transfer microorganisms from a master plate to a number of further plates in order to classify bacterial colonies
Suggest an advantage of fluorescent markers over antibiotic resistant
- obtain results quickly and easily (does not require replica plating)
- antibiotic destroys cells containing required gene
Transformation Efficiency
total of transformed colonies/mass of DNA available
Explain how sticky ends join up
- unpaired bases
- join by complementary base pairing
Recombinant DNA
sections of DNA from more than one TYPE of organism/ species
Suggest how cells can be identified using antibiotic resistance marker genes
- expose to antibiotic
- only resistant survive
Gene Therapy
- introduction of healthy genes
- replacement or inactivation of defective genes
Suggest what kinds of diseases are not suitable for gene therapy
- involve several gene
- affected by environmental factors
Suggest how viral vectors are modified
- remove/ inactivate genes which allow them to replicate
- cannot infect non-target cells
In vitro / Ex vivo
cells modified outside the body and transplanted back
In vivo
cells modified while still inside the body using vectors which transfer desired gene
Advantages of gene therapy in livestock farming
- large scale manufacture of proteins to treat disease / drugs
- decrease livestock disease
- improved efficiency would reduce environmental impact of farming
- ability to produce alternatives to petroleum based products
Disadvantages of gene therapy in livestock farming
- potential to cause suffering due to random nature of gene insertion so cannot control expression/ modifying embryos can result in birth defects
- retrovirus use can result in creation of new viruses
- low success rate
Advantages of gene therapy in agriculture
- improves quality of crops
- higher crop yield since less susceptible to disease and pest
- reduced need for pesticides so no pollution = better for environment
Disadvantages of gene therapy in agriculture
- potentially hazardous effects of uncontrolled cross breeding with wild type crops
- overuse could result in resistant weeds / pests
- potential to negatively effect animal populations / food chain
Advantages of gene therapy in medicine
- ability to replace defective cells and eradicate disease
- better alternative to current treatments such as blood/bone marrow transplants which has risk of rejection etc.
Disadvantages of gene therapy in medicine
- low success rate due to random nature of gene insertion
- potentially harmful side effects
- against religious, cultural beliefs - ‘playing God’
- unknown long term effects of modifying human genome
Polymerase Chain Reaction (In Vitro Cloning)
technique use to amplify (copy) DNA fragments
Describe how DNA is amplified by in vitro cloning
- heat DNA template to 95 degrees C
- DNA strands denature and separate (H bonds break)
- cooled to 55 degrees C to allow primers to anneal by complementary base pairing to DNA template
- free nucleotides bind to DNA template
- increase temperature to 72 degrees C for DNA polymerisation using Taq polymerase (optimum)
- cycle repeats several times
Suggest why enzymes used in PCR are obtained from bacteria found in hot springs
will not denature at high temperatures
Primer
short sequences of bases complementary to one end of DNA
Suggest why primers are used in PCR
- provide starting sequences for Taq polymerase to bind to
- prevent separate strands of DNA from re-joining
Evaluate in vitro cloning
- extremely rapid at amplifying small amounts of DNA
- do not require living cells so no culturing needed which takes time and effort
- contaminant DNA multiplied
- inaccurate
Evaluate in vivo cloning
- living cells need to cultured which takes time and effort
- many steps involved
- no risk of contamination since gene cut by same restriction endonuclease
- very accurate since mutations are rare and there are mechanisms to correct errors
- only specific gene cloned not entire sample
Describe the effect of contaminated DNA in a sample prior to PCR
- contaminant DNA amplified
- gives false result
Suggest situations where only a very small amount of DNA samples may be available for sampling and PCR can be used
- crime scene
- extinct organism
- archaeological site
Explain why individuals treated with gene therapy do not pass new gene onto their offspring
gene is not present in gametes
Evaluate the use of viral vectors
- target specific cell types
- can enter/inject DNA into cells
- RNA must be converted to DNA
- can cause immune response
Vector
- carries foreign DNA into host cell
- no benefit to carrier
DNA probe
- short, single stranded strands of DNA
- complementary to gene of interest
- radioactively or fluorescently labelled
DNA hybridisation
- section of DNA or RNA
- combines with complementary single strand of DNA
Describe the process of genetic screening
- sequence of nucleotides determined by genetic sequencing or from genetic library
- complementary DNA probe synthesised which is fluorescently labelled
- PCR to amplify DNA probes
- probe added to DNA fragments
- DNA hybridisation where probe binds
- detected using specific wavelength of light
Give applications of genetic screening
- diagnose disease
- genetic factors which increase chances of disease
- personalised medicine to determine correct dosages
- genetic factors which could be passed onto children
Evaluate genetic screening of embryos
- parents can terminate pregnancy
- parents can prepare for needs of child with disorder
- playing God by selecting desirable traits
- risk to embryo
Evaluate genetic screening of adults
- early diagnosis so higher success rate for treatment
- take precautions to prevent onset of disease
- decide if treatments will be successful to save costs
- determine dosages to avoid over prescription
Gel Electrophoresis
separating DNA fragments according to size and charge to create a genetic fingerprint
Suggest why DNA moves towards positive terminal
negatively charged due to phosphate backbone
Suggest importance of tracking dye used in gel electrophoresis
- DNA becomes visible
- current is switched off before DNA runs off end of gel
Describe the process of genetic fingerprinting
- extraction of DNA and amplify via PCR
- cut into fragments using restriction endonuclease
- separate DNA fragments by (gel) electrophoresis
- treat with alkali to split double helix to single strands
- transfer DNA fragments from gel onto nylon membrane (southern blotting)
- DNA hybridised by adding radioactive DNA probes
- autoradiograph produced from radioactive decay of probes detected by x-ray film
Suggest why DNA fragments are digested by same restriction endonucleases before gel electrophoresis
- same restriction endonuclease used to cut at same restriction sites
- produces small fragments which can be separated (long strands would take too long to separate)
Suggest why DNA fragments are submerged in an alkaline solution during gel electrophoresis
- separate double strands of DNA into single strands
- allows DNA probes to bind
Suggest why genetic fingerprints can be used to identify individuals
- variable number tandem repeats/ mini satellites
- all individuals have different VNTRs/ probability two individuals having same VNTRs is very low
- size of DNA is different so unique genetic fingerprint
Variable Number Tandem Repeats
repetitive, non-coding bases of DNA
Give applications of genetic fingerprinting
- paternity testing
- measuring genetic diversity
- forensic science
- proving pedigree for animal breed
Suggest why genetically modified embryos may not survive
- insertion of gene damages DNA / expression
- foreign antigens attacked by immune system
Suggest why electrophoresis of DNA from an individual may show one band rather than two
Homozygous
Suggest how electrophoresis could be used to estimate the number of base pairs in the separated fragments
- compare distance moved by bands with known lengths / sizes
- smaller fragments move further
Suggest how scientists could determine if DNA has been fully digested by adding together lengths of DNA fragments
- sum of strands is more than original length of DNA
- indicated undigested strand is added to total so not completely digested
Suggest reasons PCR cycle may level out
- nucleotides/ primers exhausted
- enzymes denature
DNA polymerase
Joins nucleotides
Suggest why bacteria are used over tissue cultures to produce genetically modified products
- fast replication so large production in short time
- easy to culture and extract product from culture
- bacteria are less prone to infection / contamination
Suggest why animals are used over bacteria in the production of some genetically modified products
- bacteria only survive for a short time
- animals can survive for years
Suggest how a human gene is usually obtained in gene technology
- mRNA extracted from human cells
- treated with reverse transcriptase to produce cDNA
Suggest advantages of using bacteria to synthesise human gene products
- reproduce rapidly to give a large amount of product in a short time
- product can be extracted easily from culture medium
Suggest ethical considerations for gene technology in crops
- duty to feed whole population
- risk to food chain / ecosystem
- resistant weeds/ insects could be harmful to humans
Suggest how scientists could identify the which gene produces a particular protein
- identify sequence of amino acids in protein
- use genetic code to identify the codons/base sequences
- make complementary radioactive gene probe
- to find those codons in the DNA
Suggest why restriction enzymes cut at palindromic sequences
- same sequence in opposite directions at binding site
- restriction enzyme cuts both strands at SAME site
Suggest why bioinformatics of fossils is important
- use database to compare common genes in contemporary and extinct organisms
- understand evolutionary relationships
- predict functions of genes
Suggest why it is most difficult to translate the genome of more complex organisms into the proteome
- introns (non coding DNA)
- regulatory genes
