Macromolecules: Human Manipulation Flashcards
DNA Profiling (DNA Comparison Technique)
Restriction enzymes cut DNA strands at same sequence of nucleotides - if DNA segments similar, bases will be mostly complementary
DNA Sequencing (DNA Comparison)
sequences of bases compared of two species to see how closely they match (one segment of DNA)
DNA Hybridisation (DNA Comparison)
- DNA double helix strands from species A & B heated to separate complementary strands
- strands mixed, cooled to rejoin due to complementarity
- similar species will have closely matched strands, bond more strongly
- DNA reheated, closely related DNA of species will take longer/more heat to separate… poor matches will separate quickly & at low temperatures.
* cheap, easy, less precise
Genetic Manipulation: Extraction of DNA
- break up cells
- separate nuclei (centrifuging)
- chemicals to remove nuclear membrane
- isolate DNA from nuclear proteins
- use restriction enzymes (found in bacteria) to pinpoint particular segments (4-8 base pairs), “sticky ends” ZZ “blunt ends” |
Genetic Manipulation: Identifying/Isolating Genes
two methods:
- DNA & RNA probes
- antibody method
DNA/RNA probes
- base sequence of target gene must be known
- short segments of single-stranded DNA with complementary sequence of target gene selected, radioactively labelled & mixed with double stranded DNA fragments containing target gene.
- solution heated, DNA strands separate
- cooled, some probes bind to DNA fragment/gene
- probe locates & identifies target gene
Antibody Method
- protein which target gene codes for injected into animal
- animal produces complementary antibodies (foreign)
- antibodies collected & labelled radioactively/chemically
- all of cell DNA cut into fragments, 1 or 2 inserted into large number of bacteria using recombinant DNA technology
- bacteria incubated, form colonies
- all colonies treated with labelled antibodies - will only bind to colony producing desired protein … this fragment must contain the gene
Gene Cloning
- identified/isolated genes can be cloned
- DNA & bacterial plasmid (small circular loop of DNA separate from chromosome) extracted
- restriction enzymes cut fragment of DNA containing target gene, same restriction enzymes cut plasmid DNA (same sticky ends)
- DNA fragments mixed with plasmids
- DNA ligase joins DNA fragment & plasmid to reform loop (now recombinant plasmid)
- recombinant plasmid reinserted into bacteria
- bacteria self-replicates with target gene, grows into colony
- used to produce large quantites of desired proteins (insulin, growth hormone)
Transgenesis
= incorporating foreign DNA into other organisms’ DNA
- microinjection
- vectors (bacterial plasmids, viruses)
eg. transgenic goat produced milk with human tissue plasminogen activator (TPA), protein extracted, used to dissolve blood clots in coronary arteries to reduce heart attack risk.
transgenic cotton plants produce natural insecticide, less prone to insect attacks
Polymerase Chain Reactions (PCR)
used to amplify minute amounts of DNA so that they can be confidently sequenced
(esp. in pre-natal diagnoses of genetic disease, forensic)
1. DNA sample heated to separate. DNA primer added (small segments of DNA) to stop strands from rejoining.
2. DNA polymerase & free nucleotides added.
3. DNA cooled, DNA polymerase joins nucleotides by complementary base pairingss
4. process repeated many times to obtain more strands (nucleotides added)
DNA Fingerprinting (Analysed Segments)
VNTR (Variable Number Tandem Repeats - centromere)
STR (Short Tandem Repeats/Microsatellites - scattered through genome, 2-8 bases repeated sequence)
RFLP (Restriction Fragment Length Polymorphism - fragments containing STR)
VNTR & STR are part of “junk” DNA - mutations not harmful, STR replaced VNTR due to faster analysis & greater certainty
DNA Fingerprinting (Gel Electrophoresis)
- DNA sample (hair, skin, blood, urine)
- STR fragments identified & removed using restriction enzymes
- STR multiplied using PCR
- DNA samples containing STR transferred into a well or tubes onto a gel electrophoresis plate, electric current passed through
- DNA is negatively charged…. STR fragments move toward positive terminal, speed determined by size (smaller move faster), form unique banding pattern
- banding patterns compared with DNA sample to match it
Electropherogram
- DNA samples containing STR replicated with PCR
- put into capillary electrophoresis tube
- each DNA fragment passes through laser beam, computer generates graph with peaks for each STR
- peaks compared with other DNA samples (peak = length)
Uses of DNA Profiling
- identify criminals
- custody disputes/determining genetic relationships
- detect viral DNA in the blood
- genetic defects in human eggs/embryos
- genetic faults (Huntington’s)
- identify & classify ancient, long-extinct species from fossils.
Tools of Genetic Engineering
- restriction enzymes
- DNA/RNA probes
- DNA ligase (process of ligation/”genetic glue”/reverse of restriction enzymes - ligated DNA of different species is recombinant), two matching sticky ends combine by base pairing = annealing, backbone joined by DNA ligase.
- plasmids
- viruses/viral vectors (extremely small, non-cellular particles, DNA/RNA surrounded by protein coat, able to inject genetic material into host cell causing it to manufacture viral proteins)
- microinjection (genetic material injected into recipient cell using very small fine glass needle)
