Manipulating genomes Flashcards
Define genome
The whole of an organism’s genetic information
Define genome size
Total length of DNA in a genome
Define DNA sequencing
Process of determining precise order of nucleotides in DNA
Describe Gene Sequencing Technology
- Method used for finding the base sequence of genes
- Allows comparisons of genetic variation to be made
What are terminator bases?
- Bases that when added to a DNA chain during DNA synthesis terminate it.
- Lack -OH group necessary for forming phosphodiester bond
- Prevent further elongation of a nucleotide chain
- Exist for each DNA base (i.e. ddATP, ddTTP, ddCTP, ddGTP)
Explain how the Sanger technique for DNA sequencing works
- Copies of DNA to be sequenced placed in 4 test tubes containing DNA polymerase, primers and DNA nucleotides.
- One fluorescent tagged terminator base added to each of the 4 test tubes.
- DNA polymerase replicates until terminator base is incorporated into new strand.
- DNA replicated by polymerase chain reaction (PCR) process
- Results in many strands of different lengths
- Replicated DNA run through gel electrophoresis
- Separates DNA by length
Why does a genome need to be fragmented before sequencing?
- Genome is very large
- Fewer errors with small fragments
What is the role of PCR in the sequencing of a genome?
To amplify number of copies of DNA
- Of a range of different lengths
What is the role of electrophoresis in the sequencing of a genome?
- To put DNA pieces in size order
- To read base sequence
What is the role of restriction enzymes in the sequencing of a genome?
- To cut genome DNA into smaller fragments
- To cut vectors for gene library
Explain how DNA sequencing allows the sequence of amino acids in a polypeptide to be predicted
- Sequence of bases codes for order of amino acids
- Each codon codes for one amino acid
Describe how modern high-throughput sequencing works
- Millions of DNA fragments attached to a ‘flow cell’
- DNA replicated by polymerase chain reaction (PCR)
- Fluorescent terminator bases used to stop reaction
- Images processed by computers
Explain why modern next generation sequencing is much faster than the original Sanger method
Original techniques
- Each stage carried out by hand in laboratory
Modern techniques
- Carried out in machines
- Many DNA fragments processed at once
- Much faster and more efficient
Describe the possible uses of DNA sequencing
- Synthetic biology
- Genome-wide comparisons - to better understand genetic disease
- Track disease spread and antibiotic resistance
- Identify new species
- Identify evolutionary relationships
- Understand how DNA codes for proteins
Why are models used by scientists?
- To predict what will happen in different circumstances
- To produce simpler representations of biological processes
What was the purpose of the human genome project?
To find out the entire base sequence of humans
Outline outcomes of the human genome project
- Complete human DNA sequenced
- Identification of all human genes
- Knowledge of location of human genes
- Found evidence for evolutionary relationships between humans and other animals
- Found mutations in genes causing diseases
- Developed new drugs and treatments based on base sequences
- Tailor medication to individual genetic variation
Explain how genome sequencing is used to identify the source of a disease outbreak
- Each strain of a pathogen has a different genome
- Can be accurately identified by DNA sequencing
- Place of pathogen origin can be identified
- Individuals with disease can be identified/quarantined
- Spread of strain of disease can be tracked and transmission methods understood to
prevent further spread
Explain how genome sequencing can be used to prevent antibiotic resistance
- Genome sequencing used to identify antibiotic-resistant of bacteria
- Antibiotics only used when they will be effective
- Prevents spread of antibiotic resistance
- Transmission of antibiotic-resistant bacteria can be tracked
How can genome sequencing help in the development of drugs and vaccines?
- Identify regions in pathogen genomes that may be targets
- Drugs and vaccines specific for targets can be produced
Describe how genome sequencing helps identify new species
- Compares genome to a standard sequence for the species
- Small section of conserved DNA chosen to study (DNA barcode)
- e.g. in animals: cytochrome c gene in mitochondrial DNA
- Small enough to be sequenced quickly and cheaply
- Varies enough to give clear difference between species
How has DNA sequencing changed how species are identified?
- Traditionally species identification done by observation of anatomical and physiological
features - DNA sequencing - genome similarities are examined and comparisons made to standard
species genome - More accurate but harder to carry out in the field
How is DNA sequencing used to determine evolutionary relationships?
- Looks at difference in number of mutations between species
- Average mutation rate calculated
- Used to determine when two species diverged
Define proteomics
- Study and sequencing of an organism’s amino acid sequence and protein complement
- Shows relationship between genotype and phenotype
Define intron
- Region of DNA/mRNA that does not code for a polypeptide
- Removed from pre-mRNA before it leaves nucleus (splicing)
Define exon
Region of DNA/mRNA that codes for a polypeptide
Define spliceosome
Enzyme that joins exons together
Define DNA profiling
Producing an image of the DNA of an individual
What is DNA profiling used for?
- Forensics - working out the perpetrator of a crime
- Obtaining paternity - working out the identity of the father of a child using DNA from child,
mother and possible father(s) - Analysis of disease risk - identify genes that are precursors for genetic diseases
What are short tandem repeats (STRs)?
- Repeating sections of DNA bases found within non-coding sections of DNA
- STR lengths are highly variable between individuals
Define restriction endonuclease
- Enzyme that cuts DNA at specific base sequence
How can STRs be used in DNA profiling?
- Cut STRs using restriction enzymes
- Separate using gel electrophoresis
- Fragment lengths will vary between individuals, giving unique DNA profile
What is PCR?
Polymerase Chain Reaction
- Used to amplify small amounts of DNA
What is Taq DNA polymerase?
Heat tolerant polymerase enzyme
What are primers?
Short complementary sequences of nucleotides needed to start DNA synthesis
Describe the polymerase chain reaction (PCR), including the role of Taq DNA polymerase
1. DNA heated to between 90 and 95°C
- Breaks hydrogen bonds and separates DNA strands
2. DNA cooled to between 55 and 60°C
- Allows primers to bind to target DNA sequences
- Complementary primers added
- Complementary to target sequences at ends of region to be amplified
3. Heated to between 72 and 75°C
- Taq DNA polymerase added
- Replicates the region of DNA to be amplified using complementary nucleotides
- Two strands of target DNA formed
Repeated cycles of heating and cooling amplify the target region of DNA
Why is Taq DNA polymerase used for PCR?
Thermostable
- Does not denature at 95°C during DNA strand separation
- PCR can be cycled repeatedly without stopping
What is gel electrophoresis?
Method used to separate proteins or fragments of DNA according to size
Summarise gel electrophoresis
- DNA fragments are negatively charged
- DNA placed on a gel which sieves the mixture
- Buffer solution added to carry current
- Electric current passed through gel
- DNA attracted to positive electrode (anode)
- DNA moves through gel
- Distance moved depends on mass of DNA fragment
- Smallest fragments travel fastest
- Radioactive / fluorescent tags used to visualise bands
Describe the differences between electrophoresis and the process of thin layer chromatography
TLC
- Separates by solubility
- Separates non-charged particles
- Electricity not used
- Dyes used
Electrophoresis
- Separates by size
- Separates charged particles
- Electricity used
- Radioactive / fluorescent tags used
Outline the process of DNA profiling
- DNA extraction
- DNA amplification
- Using polymerase chain reaction (PCR)
- Digestion of sample
- Using restriction endonucleases
- Separation of DNA fragments
- Using electrophoresis
- Hybridisation
- Tagging with fluorescent/radioactive DNA probes
- Development
- X-ray/UV images of the DNA profile
What is a genetically modified organism (GMO)?
Organism that has received genes from another species
Define genetic engineering
- Genes for desirable characteristics isolated in one organism
- Transferred into another organism
- Using a suitable vector
State the role of a vector
- Transfers genetic material into a cell
- e.g. plasmid, bacteriophage
Define recombinant plasmid
Plasmid containing genes from another organism
State the role of DNA ligase
- Forms phosphodiester bonds
- Joins sugar-phosphate backbones of gene and plasmid
Outline a technique used for genetic engineering
- Plasmid is used for gene transfer
- Plasmid is a small circle of DNA found in bacteria
- Restriction enzyme cuts DNA of plasmid
- Each restriction enzyme creates sticky ends
- Same restriction enzyme used to cut DNA with desired gene
- Sticky ends join gene and plasmid
- Through complementary base pairing and hydrogen bonding
- DNA ligase used to join (splice) together ends
- Recombinant plasmids formed
- Recombinant plasmids inserted into host bacterial cell
How is the take up of plasmids by bacteria improved?
Calcium ions and heat shock treatment
- Make membrane permeable to plasmid
Electroporation
- Electric current makes membrane porous to plasmids
Explain how reverse transcriptase can also be used to isolate a desired gene
- mRNA for desired gene isolated
- Reverse transcriptase added
- Produces single strand of complementary DNA (cDNA)
Explain how bacteria that have been successfully transformed are identified
- Plasmids used contain marker gene
- e.g. for antibiotic resistance
- Used to determine whether plasmid has been taken up successfully
- Second marker gene also engineered in
- Usually fluorescence or enzyme which changes colour of medium
- Required gene inserted in middle of second marker
- Bacteria showing marker trait are not successfully engineered
Describe how genetic modification has been used to produce human insulin at an industrial scale
- Gene coding for human insulin removed using restriction endonuclease
- Gene has sticky ends
- Gene amplified using PCR
- Bacterial plasmids cut open using same restriction endonuclease
- Plasmids and insulin gene have complementary sticky ends
- Plasmids are acting as a vector
- Plasmid and insulin DNA are mixed
- Sticky ends fuse due to complementary base pairing
- DNA ligase fuses gene and plasmid by forming sugar-phosphate bonds in DNA
- Forms ‘recombinant plasmids’
- Recombinant plasmids are mixed with E. coli
- E. coli absorb recombinant plasmids
- Take up improved by addition of Ca2+ and heat shock
- Or electroporation
- E. coli grown in a fermenter
- Human insulin extracted
What is a bacteriophage?
- Virus that infects bacteria
- Can be used to insert foreign DNA into bacteria
Name a vector used to introduce foreign DNA into plant cells
Agrobacterium tumefaciens
Define gene therapy
- Replacement of mutant, faulty allele with healthy allele
- To treat genetic diseases
Define somatic cells
Body cells (i.e. not sex cells)
Define somatic cell gene therapy
- Mutant allele replaced with healthy allele
- In all affected somatic cells
Define germ cell
Gamete/sex cell
Define germ line cell gene therapy
- Healthy allele inserted into germ cells (usually egg cells)
- Or zygote immediately after fertilisation
- Individual born with healthy allele
- Allele passed onto offspring
What are the disadvantages of somatic stem cell therapy?
- Somatic cells have limited life-span
- Replacement cells from stem cells will not contain healthy allele
- Healthy allele not passed on to offspring
Outline the ethical concerns about germ line cell gene therapy
- Impact of inserting genes into germ cell is unknown
- Human rights of the unborn individual could be violated
- Could eventually be used for choosing desirable characteristics in offspring
Give potential uses of genetically modified microorganisms
- Produce human hormones (e.g. insulin)
- Store DNA as DNA library
- Developing novel medical treatments and industrial processes
Why is there less ethical debate about the use of microorganisms in genetic engineering?
- Have been used safely for many years
- Produce many beneficial materials so benefits are very tangible
- e.g. insulin, antibiotics
- No welfare issues
Define pharming
Genetically engineering animals to produce human medicines
Describe the process of genetically modifying animals
- Copy of human gene coding for desired protein isolated or synthesised
- Gene introduced into genetic material of fertilised cow, sheep, or goat egg cell
- Promoter sequence added to ensure gene is expressed only in mammary glands
- Fertilised transgenic embryo returned to mother or surrogate to grow to birth
- When mature transgenic animal conceives and gives birth, it produces milk containing
desired human protein to be harvested
Advantages on in vitro gene cloning
Advantages of PCR
- Quicker
- No bacterial growth or screening stages
- Uses less equipment
- Uses less space
- In vivo requires many plates to be stored
- Less labour-intensive
- PCR set to run and left
- Combines selection of gene and amplification - in vivo requires separate steps
- Primer selects only correct gene to be copied
- In vivo needs probe to identify correct gene
- Safer
- PCR does not use whole cells which could cause contamination
- Can use lower quality DNA
Advantages of in vivo cloning
- Less prone to mutation
- Taq polymerase occasionally inserts wrong base in PCR
- Early mutation reproduced many times in PCR
- Less expensive
- Materials for growing bacteria cheap
- Less technically complex
- Useful when gene less well known
- e.g. when searching for new gene
Where do restriction enzymes cut DNA?
At palindromic sequences
Define bioinformatics
Development of software needed to organise and analyse raw data
Define computational biology
- Uses data from bioinformatics
- To build theoretical models of biological systems
Explain the impact of bioinformatics and computational biology on the usefulness of DNA sequencing
Bioinformatics
- Allows scientists to analyse large amounts of data generated during sequencing of billions of base pairs in genomes
- Helps identify patterns
Computational biology
- Uses results to build up models
- e.g. the spread of disease, the evolutionary relationships between organisms
- Uses models to determine possibilities in different circumstances
Outline the role of bioinformatics in the genetic modification of plants
- Bioinformatics allows rapid analysis of large numbers of samples
- Target genes can be identified rapidly
- Sequences that control regulation of target gene also identified
Outline how DNA sequencing and bioinformatics could be used to increase the effectiveness of a virus vaccination programme
Sequencing
- High mutation rate means many strains of virus exist
- Can identify viral strains
- So vaccine contains correct antigen
Bioinformatics
- Facilitates access to large amount of data
- Can be shared worldwide
- Can identify source of outbreak
- Can identify vulnerable populations
- Vaccination program can target certain areas or individuals
Define synthetic biology
The design and construction of artificial biological pathways or organisms
How can synthetic biology be used?
Genetic engineering
- Change of biological pathway or genetic sequence
Industrial processes
- e.g. immobilise enzymes to increases efficiency
- e.g. production of drugs from microorganisms
Synthesis of new genes
- Able to replace faulty genes
- e.g. as potential treatment fro cystic fibrosis
Define pharming
Genetically engineering animals to produce human medicines
Describe the process of genetically modifying animals
- Copy of human gene coding for desired protein isolated or synthesised
- Gene introduced into genetic material of fertilised cow, sheep, or goat egg cell
- Promoter sequence added to ensure gene is expressed only in mammary glands
- Fertilised transgenic embryo returned to mother or surrogate to grow to birth
- When a mature transgenic animal conceives and gives birth, it produces milk containing
desired human protein to be harvested
Compare somatic cell gene therapy and germ line cell therapy
Somatic gene cell therapy is replacement of mutant gene in body cells with healthy allele, whereas germline cell therapy is replacement of mutant gene in egg, sperm or early embryo with healthy allele.
In somatic cell gene therapy, healthy, allele not passed on to offspring, whereas in germline cell therapy, healthy allele will be passed on to offspring.
