manipulating genome : profiling Flashcards
1
Q
producing a DNA profile
A
- DNA extracted from a tissue sample (PCR)
- restriction endonucleases cut the DNA into fragments : cut DNA at a specific nucleotide sequence = restriction site
- makes two cuts , one through each double helix strand
- fragments are separated using gel electrophoresis
- DNA fragments are transferred from gel to nylon membrane by southern blotting
- DNA probes are added to label the fragments
- radioactive probes attach to specific fragments
- membrane with radioactively labelled DNA fragments is placed onto an x-ray film
- X ray film reveals dark bands where the
radioactive/fluorescent DNA probes have attached
2
Q
PCR
A
- DNA is replicated
- temp in PCR = increased to 90-95 for 30s
- denatures DNA
- breaks H-bonds holding DNA strands so they separate
- temp decreased to 55-60
- primers bind to ends of DNA strands
- temp inc to 72-75 for at least a min
- ^optimum temp for DNA polymerase
- DNA P adds bases to the primer - builds complementary strands of DNA
- produces double stranded DNA identical to original
- enzyme Taq polymerase is used
- obtained from thermophilic bacteria in hot springs
3
Q
uses of DNA profiling
A
- forensic science
- PCR + DNAP is done on traces of DNA left
- obtained from blood, semen + saliva
- provides evidence for a crime
- proves paternity of a child
- identifies at risk individuals of disease
4
Q
electrophoresis
A
- DNA sample loaded into wells of agarose gel
- loading dye added if not radioactively/fluorescently labelled
- gels covered in a buffer sol
- electrodes connected a either end
- DNA = -ve moves through gel away from cathode
- gel provides a resistance force
- smaller DNA fragments move further in time provided
- smaller fragments at bottom whereas larger remain closer to cathode
- proteins are mixed with a chemical that denatures them so they all have the same charge
5
Q
DNA sequencing
A
- process of determining the order of nucleotides within a DNA mol
6
Q
high throughput sequencing
A
- sequence faster
- less cost
- section of DNA into fragments
- split into single strands
- strand from each fragment attached to a small bead
- PCR amplifies the fragments
- each bead on a separate well
- free NT added to wells attach to DNA strands
- CBP
- wells have specific enzymes
- allow light emitted when bases added to DNA
- computers analyse the occurrence and intensities of light emitted
- interprets the DNA sequence
7
Q
how gene sequencing has allowed for
genome-wide comparisons between
individuals and between species
A
- computers analyse + compare genomes
- reveals patterns in the DNA we inherit
- diseases we are vulnerable to
- sequencing genomes of pathogens inc bacteria/virus’
- scientists can track progress of an outbreak / doc find out source of an infection
- understand evolutionary relationships
8
Q
gene seq : amino acids in polypeptides to
be predicted
A
- sequencing gene = sequence of amino acid + primary structure can be predicted
- developed area of ‘synthetic bio’ = create biological mol
9
Q
synthetic biology
A
- includes diff techniques:
- genetic engineering
- synthesis of new genes to replace faulty ones
- synthesis of a new organism
- redesigning biological systems to perform better and include new mol
10
Q
genetic engineering : isolating the gene
A
- use restriction endonucleases to cut required gene from DNA
- one strand is longer
- regions with unpaired , exposed bases = sticky ends
- sticky ends make it easier to insert the desired gene into DNA
OR - isolate mRNA for desired gene
- use enzyme reverse transcriptase to produce single strand of complementary DNA
- easier to identify desired gene
11
Q
inserting DNA into a vector
A
- e.g bacterial plasmid
- DNA fragment inserted into vector DNA
- vector DNA cut open using same R.E enzyme in DNA isolation
- sticky ends of vector completer to sticky ends of DNA fragment
- vector DNA + DNA fragment are mixed with DNA ligase
- DNA ligase forms phosphodiester bonds
- sugar phosphate backbone
reformed- process = ligation - vector DNA + DNA fragment = recombinant DNA
12
Q
electroporation
A
- electrical current applied to bacteria
- membrane = more porous
- plasmid moves into cells
13
Q
benefits/risks of genetic manipulation
A
- ethical : uncomfortable with inserting human genes into microorganisms
- GM of plants will help feed growing population
- overcome env issues
- insect resistance in soya beans : inserted gene into soya to make Bt protein which is toxic to many pest insects that attack plants. Allows for higher yield with less labour + expense
- insects might become resistant
- reduced biodiversity if herbicides overused to destroy weeds
- fear of superweeds
14
Q
somatic cell gene therapy
A
- replacing mutant allele with a healthy allele in the affected somatic body cell
- not a cure
- reapplied to many cells
- uses viral vectors
- sucessful treatment for leukemia
- temp solution
- still pass on faulty genes to children
15
Q
germ line cell gene therapy
A
- healthy allele inserted into germ cells (eggs)
- or into embryo straight after fertilisation
- individual is born healthy with normal allele
- healthy allele passed onto offspring
