25 Gene Technologies Flashcards
what is the function of PCR?
{polymerase chain reaction}
artificial DNA replication
what are the four items required for PCR?
- the DNA fragment being copied
- free phosphorylated nucleotides - to synthesise new DNA strand
- Taq. DNA polymerase - to bind nucleotides together
- primers - provide starting sequence for Taq. DNA polymerase
what is the name and function of the computer used in PCR?
a thermocycler
varies temperature at precise time intervals
PCR: strand separation (stage 1)
target DNA heated to 95.C for 5 mins –> separated into 2 single strand lengths
PCR: primer binding (stage 2)
solution cooled to 55.C
primers H bond to complementary base sequences on single stranded DNA
PCR: strand synthesis (stage 3)
heated to 72.C (optimum for Taq. DNA polymerase)
free complementary nucleotides joined to DNA strands, beginning at the primers –> 2 identical strands produced from original
PCR v. semi-conservative DNA replication
+ one old, one new strand in each molecule of DNA
+ free nucleotides
- PCR produces only short strands ; whole chromosomes copied in S-C R
- PCR requires primers
- PCR requires high temperatures to separate DNA ; S-C R uses DNA helicase
what is the function of electrophoresis?
preparation and analysis of DNA for sequencing
what are the three items required for electrophoresis?
- gel plate containing agarose, and covered by electrolyte buffer
- electrodes
- DNA strands
how are samples of DNA obtained in electrophoresis?
restriction endonucleases cut DNA into fragments
how do the DNA fragments separate during electrophoresis?
placed in wells at cathode end of tray
current passed through for ~2 hours
phosphate groups are attracted to the anode
shorter fragments of DNA move further and faster
how are the DNA bands visualised following electrophoresis?
using fluorescent dye and UV light
electrophoresis v. chromatography
+ involves the displacement of different molecules within a medium for identification
+ separates molecules according to size
+ some molecules slowed more by stationary phase than others
what are haplotypes?
sets of genes inherited together from one parents
what types of DNA are used when identifying haplotypes? why?
mitochondrial and Y-chromosomal DNA
neither undergo recomibination
what are single nucleotide polymorphims (SNPs)?
sequences of DNA that vary between individuals by a single nucleotide
what is a modern use of identifying SNPs?
give and indication of susceptibility to disease
and responses to drugs/chemicals/vaccinations
(can also track inheritance)
what are variable number tandem repeats (VNTRs)?
patterns of repeated adjacent nucleotides
what is a modern day use of VNTRs?
use as genetic fingerprint
can be used in electrophoresis –> forensic analysis
what is the definition of genetic engineering?
the manipulation of an organism’s DNA
what is the differenced between ‘transformed’ and ‘transgenic’ organisms?
transformed = organisms with recombinant DNA
transgenic = organisms which have been GE to include a gene from a different species
how do restriction enzymes (endonucleases) isolate a DNA fragment?
enzyme has specific recognition sites where it digests DNA
some produce a staggered cut (‘sticky ends’), which can anneal to other complementary sequences
how is a DNA fragment inserted into a vector? (4 stages)
DNA ligase cuts vector DNA, then anneals plasmid and recombinant gene
bacteriophages are used to inject DNA into host cell
ice cold CaCl(2) encourages bacteria to take DNA up by increasing permeability of bacterial cell wall
plasmids added + mixture heat shocked to 45.C for 1-2 mins
how is a gene produced from an mRNA template? (4 stages)
reverse transcriptase synthesises a single strand of cDNA complementary to mature mRNA
DNA polymerase used to join free nucleotides together to form second DNA strand
plasmid vector opened with restriction enzyme
gene placed in plasmid with DNA ligase
why are reporter genes required in genetic engineering?
to identify which bacterial cells have taken up the recombinant DNA
how do reporter genes work?
desired gene inserted into reporter gene sequence so that it does not work
cells not displaying reporter property (e.g. fluorescence) have taken up gene
how are bacteria encouraged to take up recombinant DNA?
ice cold CaCl(2) - increases permeability of cell wall
growth on agar plates + replica plating
describe the benefits of genetically engineering ‘Enviropig’
mouse + E. coli DNA prompts phytase production in pig’s salivary gland
phytase breaks down phosphorous into phosphates –> can be absorbed
∴ less phosphorous contaminates human water supply
give an example of a genetically engineered crop plant
Agrobacterium tumefaciens
a naturally occurring soil bacterium which can introduce new genetic material into plant cells
infects wound sites of dicotyledonous plants, causing formation of crown gall tumours
discuss ‘knockout mice’
mice that are GE to have an inactivated gene
target gene knocked out by incorporating a nucleotide sequence resembling it into an embryonic stem cell, which is then fused with a de-nucleated embryo
used to study effect of certain genes on diseases
what is the difference between an intron and an exon?
exons code for sequences of amino acids
introns do not, and are removed from mRNA after transcription (–> mature mRNA)
what is alternative splicing?
when mRNA molecules from the same gene have different combinations of introns retained and rejoined
what is the function of alternative splicing?
allows genetic diversity
give a use of RNA interference
gene control - post-transcriptional gene splicing
laboratory tool
treatment of disease
silencing of genes in tumour cells
reducing gene activity in cell culture
what is the mechanism of RNA interference?
micro RNA (miRNA) and small interfering RNA (siRNA)
delivered from double-stranded RNA
dicer enzyme cuts double-stranded RNA into 20-25 nucleotide lengths
Argonaute protein selects and binds to one strand of RNA
siRNA binds to specific sequences of complementary nucleotides on mRNA
Argonaute protein breaks mRNA (making it non-functional)
