Manipulating Genomes - 6 Flashcards
What is PCR
Polymerase chain reaction is a method for amplifying DNA fragments
What 5 components are required for PCR
DNA fragment
Primers
DNA polymerase
Free nucleotides
Thermocycler
What are the 3 stages of PCR
Separation
Addition
DNA synthesis
What occurs during separation in PCR
Heating DNA at 95c separates hydrogen bonds between complementary base pairs
What happens during addition in PCR
Primers attach to specific starting points on each of the separated DNA strands by forming hydrogen bonds
55 degree c
What happens during DNA synthesis in PCR
DNA polymerase adds free nucleotides to the ends of the primers, extending the DNA strand to form a complete copy
72 degree c
Advantages of PCR
Rapid speed
Precision
Low DNA needed
No cells needed
What is gel electrophoresis
Technique used to separate molecules such as DNA, RNA or proteins based on size
How to set up gel electrophoresis
Insert a gel tray with solidified agarose gel into a gel tank
ensure the wells are close to the negative electrode to position the gel correctly
Pour buffer solution over the gel until it is submerged to maintain a constant pH
How to load samples in electrophoresis
Mix samples with loading dye to make them visible
Carefully deposit equal volumes of each sample into wells
Touch micropipette tip to the buffer to prevent damaging the gel
What 3 fields can DNA sequencing be used for
Bioinformatics
Computational biology
Genomics
What is bioinformatics
Involves developing software, computing tools and models to collect, store and analyse biological datasets
What is computational
Uses bioinformatics tools and biological data to model biological systems and processes
What is genomics
Applies DNA sequencing and computational biology to study genomes of organisms
What are the benefits of sequencing pathogen genomes
Identifying the sources and transmission routes of disease
Detecting antibiotic resistance
Developing new treatments
Monitoring disease outbreak
What is DNA barcoding
It involves comparing the DNA sequences of an unidentified organism to a database
Advantages of DNA barcoding
Fast and affordable sequencing
The classification of new species
Updating of classifications
Potential applications of synthetic biology
Synthesising functional genes to replace faulty ones
Utilising microorganisms to produce drugs
Constructing artificial genomes
What is recombinant DNA
DNA that contains nucleotides from two different organisms
What are the key stages in gene transfer
Desired gene is identified and isolated
Multiple copies of the gene are made using PCR
The gene is inserted into a vector
Vector delivers the gene into cells
Cells with the new gene are identified and cloned
Explain the process if reverse transcriptase
mRNA is extracted from cells
mRNA is reverse transcribed using the reverse transcriptase enzyme and DNA nucleotides
This makes a cDNA strand identical to the original DNA strand
cDNA, free nucleotides and the DNA polymerase can then form the other strand of DNA
How are restriction enzymes used to cut DNA
DNA is incubated with chosen restriction enzymes
Restriction enzymes identify palindromic sequences in the DNA double helix and cut double-stranded DNA
Recognition sequences at either end of desired fragment allow restriction enzymes to separate fragments
Enzymes cut target gene fragment out via hydrolysis
Explain how DNA is inserted into vectors
Vector is cut open at specific site using restriction enzyme
The same restriction enzyme is used to cut the target DNA fragment creating sticky ends
DNA ligase forms phosphodiester bonds between the sugar and phosphate.
Joining the sticky ends of the vector and fragment
How is recombinant DNA transferred into cells
Plasmids - Host cells are treated to enhance uptake of plasmids.
Electroporation, makes bacterial membranes more porous
Bacteriophage vectors - Inject their DNA into host bacterial cells
Advantages of GM crops
Involves less labour than traditional herbicides and pesticides.
Cheaper
Increases crop yield
Disadvantages of GM crops
Monocultures, reduces biodiversity
Can create herb resistant weed
Advantages of GM farmed animals
Improves quality
Improves quantity
Improves productivity
Advantages of pharming
Enables mass production of rare treatment
Makes drugs more accessible
Disadvantages of pharming
Raises animal welfare concerns
Advantages of GM pathogens
Offers potential treatment
Can produce essential medicine
Vaccinations
Disadvantages of GM pathogens
Risk of infections
Could be misused
What is somatic gene therapy
Replaces mutant alleles with healthy alleles in affected body cells
What is germline gene therapy
Inserting a healthy allele into germ cells or embryos to prevent genetic diseases from birth
Issues of somatic gene therapy
Very difficult to do
Effects only last a short time
Issues of germline gene therapy
Unethical
Causes irreversible changes
Could be used for non therapeutic traits
Benefits of gene therapy
Extends lives
Improves quality of life
Allows children to live without genetic disorders
Reduces overall disease burden
