8 The Control of Gene Expression- Gene Technology Flashcards
What is recombinant DNA and how is it produced?
-Where fragments of DNA are inserted into other sections of DNA
-Genetic code= universal; any section of DNA can be taken from one organism, placed inside another
-It is then transcribed & translated to produce proteins (also universal processes)
-Process of transferring sections of DNA produces recombinant DNA
What are fragments and transgenic organisms?
-The sections of DNA that are transferred
-Organism that has received fragments of DNA
Why are DNA fragments produced and what are the ways in which this happens?
-Recombinant DNA often involves inserting specific gene of interest into DNA; gene normally encodes protein w/ useful properties. Transferred gene= target gene
-To transfer target gene, it needs to be removed from DNA in section called a DNA fragment, can be produced in three ways:
-Reverse transcriptase
-Restriction endonuclease
-Using a gene machine
What can be done to DNA fragments after they have been produced?
They can be amplified either in vivo (inside organism) or in vitro (outside of organism)
What happens after a fragment has been produced?
It can be inserted into the genome to produce a transgenic organism
How can reverse transcription be used to produce DNA fragments?
-mRNA; single-stranded molecule produced when a specific length of DNA (target gene) is transcribed, it is complementary to base sequence in target gene
-mRNA sequence can be used as template for producing fragments of DNA
-Reverse transcriptase= enzyme that converts single-stranded mRNA into double-stranded DNA, can be used in producing DNA fragments by converting mRNA for target gene into double-stranded DNA
-DNA produced= complementary DNA (cDNA)
How are DNA fragments of insulin isolated from pancreatic cells?
-mRNA for insulin → isolated from pancreatic cells
-mRNA is mixed w/ reverse transcriptase
-This converts mRNA → cDNA
-cDNA can be used to produce recombinant DNA
What are recognition sequences and their function?
-Sections of DNA where base sequence has palindromic base pairs (have sequence of base pairs that are the same but in opposite directions)
-Can be used to isolate target gene if there are 2 sets of sequences either side of gene
What is the job of restriction endonucleases and how is the target gene obtained?
-Restriction endonuclease enzymes bind to recognition sequences
-Each restriction endonuclease binds to specific recognition sequence
-If 2 restriction endonucleases bind to 2 recognition sequences surrounding target gene, target gene can be cut out of DNA
How are DNA fragments produced using restriction endonucleases?
-DNA containing target gene is mixed w/ restriction endonucleases
-Restriction endonucleases bind to recognition sequences on either side of target gene
-Target gene is cut out of DNA
How can DNA fragments be produced using gene machines?
-DNA fragments can be produced by synthesising target gene sequence w/ free-floating nucleotides; useful as DNA template isn’t necessary
-Sequence for target gene—> obtained from database
-Nucleotides are added in correct order to synthesise correct base sequence
-Protecting groups added throughout synthesis to make sure correct nucleotides are added & no side branches are produced
What can be done to DNA fragments after they have been produced?
They can be amplified either in vivo (inside organism) or in vitro (outside of organism)
What are the steps involved for in vivo amplification?
-Vector= form of transport for DNA fragment. Vector DNA is cut open at specific regions (recognition sequences) by restriction endonuclease enzymes so each end has a short single-stranded section; the sticky ends
-DNA fragments have sticky ends complementary to sticky ends on vector DNA as they’ve either been cut from DNA using same restriction endonucleases/several nucleotides have been added onto ends of fragment.
-They bind together as DNA ligase enzyme attaches sticky ends together; this is called ligation
-DNA fragment has been inserted into vector DNA; this is recombinant DNA
-Vector transfers recombinant DNA to host cell. If it’s a plasmid: host cells take up recombinant DNA via heat-shock (where cells are heated at 42°C for 1m). If it’s a bacteriophage: recombinant DNA injected into host cells
-Cells successfully taken up the recombinant DNA are transformed; said to be genetically modified but not all will be (transformed cells identified using marker genes- inserted along w/ recombinant DNA & confer antibiotic resistance
-Transformed cells can be identified by placing them on agar plate w/ antibiotics; only cells that have successfully taken up recombinant DNA will be able to survive
-Transformed cells can be grown in large # to amplify target gene
What are the steps involved for in vitro amplification?
-DNA fragments are mixed w/; primers (short sections of DNA), enzyme DNA polymerase (produces new strands of DNA), free-floating nucleotides
-Together these components form reaction mixture
-Heat reaction mixture to 95°C; high heat causes hydrogen bonds between DNA strands to break & DNA to separate into 2 separate strands
-Cool reaction mixture to 65°C; causes primer to anneal to the 2 separate strands of DNA. Primers= complementary to beginning of 2 strands
-Heat reaction mixture to 72°C (optimum temperature for DNA polymerase activity)
-DNA polymerase produces 2 new DNA strands by using the two separated strands of DNA as template & adds free-floating nucleotides complementary to template strands of DNA
-Primers allow nucleotides to bind to one another, produce strand of DNA
-This process of heating, cooling & heating produces 2 new strands of DNA from 1
-Can be repeated as many times as possible to quickly amplify # of DNA fragments; doubled in each cycle of PCR
How is recombinant DNA used for genetically modified crops and why is this beneficial?
-Can be used to genetically modify crops to improve their yield
-Traits that can be improved; resistance to disease, tolerance to application of herbicides & pesticides, tolerance of adverse environmental conditions
How can recombinant DNA be used for genetically modified livestock and why is this beneficial?
-Can be used by farmers to make production of meat more economically viable
-Traits that can be improved; grow faster & larger, resistance to disease
How can recombinant DNA be used in nutrition?
Can be used to increase nutritional value of food
How can recombinant DNA be used in treating diseases?
Can be used to produce medicine and hormones to treat diseases
How can recombinant DNA be used to benefit industry?
Can be used to manufacture enzymes, which can then be used in industry
What are the ethical concerns to do with the spread of genes from recombinant DNA?
-GM crops & livestock → produced when beneficial gene is inserted into their genome to improve certain trait
-Genes could be transferred into other organisms where it’s harmful
-Genes from genetically engineered (transgenic) crops could also be spread to organic crops
What are the ethical concerns to do with the unforeseen impacts of recombinant DNA?
-Genetic modifications to an organism could have unforeseen effects & disrupt normal gene function
-Use of genetically engineered organisms could lead to ↓ in variety in populations
What are the ethical concerns to do with the economic consequences from recombinant DNA?
-Could be economic consequences for some countries if genetically engineered crops can be grown in different countries where it wasn’t previously possible
-Companies that are able to invest more money in recombinant DNA technology may out-compete others
What are the ethical concerns to do with medical uses of recombinant DNA?
Some activists are concerned that using recombinant DNA in medicine could lead to unethical uses of genetic engineering
What is gene therapy?
A genetic engineering technique used to cure disease
What is the procedure of gene therapy?
-Introduction of target gene into genome
-It’s then transcribed & translated to produce desired protein
-Protein counteracts effect of disease caused by a mutation
How does gene therapy vary based on allele interactions?
-The way that gene therapy is used depends on allele interactions of gene causing disease
-If mutation is in recessive allele; wild-type (typical of species) dominant allele inserted into genome, counteracts mutant alleles
-If mutation is in dominant allele; allele that ‘silences’ mutant allele is inserted in genome
How are vectors used in gene therapy?
-Vectors (eg plasmids, bacteriophages) are used to insert target gene into genome
-Vectors transport allow gene to be taken up by cells of host; genome is then transformed
What are the 2 types of gene therapy?
-Somatic; altering of alleles in adult body cell
-Germline; altering of alleles in sex cells; illegal in humans
What are DNA probes and what are they used her?
-A section of single-stranded DNA, complementary to the DNA of target allele
-Used to identify which alleles are present in sample of DNA; can be used in medical diagnosis by identifying if individual has specific disease-causing alleles
What is hybridisation and how does it occur?
-Complementary base sequence in probe means that if target allele is present in DNA that’s being sampled, it’ll bind to the DNA probe
-Hybridisation → when a base sequence binds to complementary base sequence
How are DNA probes used in diagnosis?
-DNA probes are labelled w/ a radioactive phosphate/ fluorescent tag
-If disease-causing allele hybridises w/ probe, label will be detected
What are the 2 techniques used to diagnose the presence/absence of disease-causing alleles in a DNA sample?
Electrophoresis
Microarrays
What is electrophoresis and how is it done?
-Technique that separates fragments of DNA according to size
-Sections of DNA → transferred onto nylon sheet
-DNA probes labelled w/ fluorescent tags are ‘washed over’ the DNA fragments
-DNA probes hybridise w/ any fragments complementary to probes
What is a microarray and how is it used in diagnosis?
-A slide w/ many indents each containing DNA probes for specific gene
-Use many DNA probes at once
-Fluorescently labelled DNA sample is ‘washed over ‘ the microarray
-Any DNA fragments complementary to probes will hybridise
How can DNA probes be identified and what do they indicate?
-DNA probes that have hybridised to DNA DNA sample in either electrophoresis/ microarray can be identified
-If probes are labelled w/ fluorescent tags, they’ll be revealed by UV light
-If probes are labelled w/ radioactive phosphate, they’ll revealed by X-ray
-Identified DNA probes indicate which alleles are present in a sample of DNA
What can genetic screening be used to identify?
-If an individual is a carrier of a genetic disease
-If an individual is at greater risk of developing a disease
-How likely an individual is to respond to a particular drug
What is genetic counselling and what is its purpose?
-Info gained from genetic screening can be used in genetic counselling
-This is used to advise patients about genetic screening, advice considers;
-If screening is advisable
-What results of screening mean
-How to prevent/ treat condition identified via screening
How can genetic screening aid personalised medicine?
-Info gained from genetic screening can be used in personalised medicine
-Genes in individual influence how likely they are to respond to a particular drug
-Genetic screening can help doctors prescribe most effective drugs for their patient
What are VNTRs and what are they used for?
-Variable number tandem repeats; sections of DNA that don’t code for any proteins
-Used in genetic fingerprinting
What is the structure of VNTRs?
-Sections of DNA found throughout genome
-Sequences of VNTRs consist of series of bases repeated in the same sequence many time
-Number of times sequence is repeated is varies; why VNTRs have their name
What is the function of VNTRs?
-# of repeats in a VNTR can vary among individuals; very unlikely that 2 individuals will have exact same # of them in the same place in genome
-This allows comparisons to be drawn between individuals
-VNTRs are used in this way in genetic fingerprinting
What is gel electrophoresis?
Technique used to compare genetic fingerprints of individuals; everyone has a different one
What are the steps involved in gel electrophoresis?
-DNA sample is extracted from individual, done by taking a swab inside someone’s mouth/blood sample
-The sample is amplified many times using PCR, which generates many copies of same sample
-DNA fragments made from PCR are labelled w/ fluorescent label; allows fragments to be identified when exposed to UV light
-The many fragments are inserted → a well in a gel covered in a buffer solution conducting electricity w/ positively charged electrode at one end & negatively charged electrode at other end. DNA is inserted at negative end of gel
-DNA = negatively charged so when electric current is passed through get it’ll move away from negative electrode towards the positive
-Smaller DNA fragments will move through gel more quickly & travel further than larger fragments
-electric current is removed after approx 10mins. DNA fragments of diff lengths in sample will have moved differing distances up gel; presence form bands of DNA, produce ‘ladder’= considered their genetic fingerprint
-The diff fragments can be identified by exposing gel to UV light as they’re fluorescently labelled
How can genetic fingerprinting be applied to genetic relationships?
-Can be used to identify genetic relationship between individuals
VNTRs are inherited from individual’s parents; offspring will have a combo of their mother & father’s genetic
-So, can be used for paternity tests
How can genetic fingerprinting be applied to genetic variability?
-This is how much genetic diversity exists in a population, can be identified using genetic fingerprinting
-The more varied the genetic fingerprints of a population are (eg fewer overlapping bands in gel electrophoresis) the greater genetic variability
How can genetic fingerprinting be applied to forensic science?
-Can be used to compare DNA at a crime scene to potential suspects
-DNA is sampled from a crime scene & fingerprint is produced in gel electrophoresis
-DNA from scene can be used to find individual w/ closely matched genetic fingerprint
How can genetic fingerprinting be applied to medical diagnosis?
-Some genetic disorders are produced from multiple mutations; may produce specific genetic fingerprint
-Disorders can be diagnosed by comparing genetic fingerprint of individual to genetic pattern of condition
How can genetic fingerprinting be applied to interbreeding?
-Inbreeding between animals & plants → problematic in agriculture as it ↓ genetic diversity (increases chance of genetic disorders & extinction)
-Can be avoided using genetic fingerprints
