3.6 - Molecular biology toolkit Flashcards
recombinant
bringing together genetic material from different sources, including different species
restriction enzymes
bacterial enzymes that cleave DNA in a highly specific manner
how do restriction enzymes recognise cleavage sites
cleavage sites (4-8 nucleotides) that are palindromic and then cleave each strand of DNA
agarose gel electrophoresis of DNA
- DNA fragments generated by restriction enzyme
- fragments visualised by staining with DNA-binding dye which fluoresces under UV light
- DNA fragments loaded into well at cathode (-ve) end, fragments through gel towards anode (+ve) when voltage applied across gel
- smaller fragments move quicker and travel further
how can PCR be used for DNA fingerprinting? (2)
- PCR capable of amplifying single molecule of DNA, can then be visualised in gel or sequenced
- used to detect mutations that cause cancer, verify presence of infectious agents and determine guilt/innocence of crime suspects
what does DNA fingerprinting make use of?
variability of non-coding “microsatellite” DNA between individuals
plasmid
small, circular double-stranded DNA molecule separate from cell’s chromosomal DNA (found naturally in bacteria)
insertion of DNA into plasmid vector (3)
- restriction enzyme digest of DNA often generates fragments with staggered/”sticky” ends
- vector can be cleaved by same restriction enzyme used to generate fragment, when fragment and cleaved vector mixed, sticky ends anneal
- DNA ligase used to covalently join fragments and vector, making recombinant DNA
reporter genes
reporter genes in vector, such as antibiotic resistance genes, make identification of vectors with inserter DNA sample easier
expression of eukaryotic gene in E. coli (2)
- expression plasmid drives transcription of cloned DNA insert to make large quantities within bacterial host cell
- host cell translates mRNA to make protein, can be purified in large quantities
what key human medicines are produced using expression of a eukaryotic gene in E. coli?
insulin - previously animal derived, harvested from cow/pig pancreas extracts
how is a genomic library made
fragmenting entire genome of an organism and inserting fragments into a vector
steps in constructing a genomic library (5)
- genomic DNA cut into fragments of few thousand base pairs using restriction enzyme
- fragments ligated (bound) into plasmid vector, plasmids transformed into E. coli bacterial host cells (each cell picks up single plasmid)
- transformed E. coli propagated on nutrient plates
- each bacterial colony division of single transformed E. coli cell (single clone)
- aim is for every sequence in genome to be represented in total library of clones
DNA sequencing
process of determining nucleic acid sequence (order of nucleotides in DNA)
preparation of cDNA from eukaryotic DNA (4)
- DNA copy of mRNA that is transcribed from active gees can also be made using RT-PCR
- total mRNA extracted from selected type of cell (or embryo tissue etc) and DNA copy made using reverse transcriptase
- RNA strand partially degraded using RNase, and complementary DNA (cDNA) strand synthesised using DNA polymerase
- allows determination of genes expressed in certain cells, number of uses
use of quantitive PCR (qPCR)
quantity of individual transcripts in cell can be determined by qPCR
quantitive PCR (5)
- mRNA isolated and converted into cDNA (complimentary DNA)
- used as target DNA in PCR reaction carried out in presence of dye that fluoresces when bound to the duplex DNA
- PCR cycle at which fluorescence becomes detectible is inversely related to original number of target templates
- DNA sequence attached to solid support in defined pattern to generate microarray
- fluorescently labelled cDNA then hybridised to microarray to reveal expression level of each gene on chip
DNA microarrays
(gene chips) - allow determination of expression pattern of large number of genes simultaneously
how can new genes be inserted into eukaryotic cells? (3)
- calcium phosphate mediated uptake
- microinjection into cells’
- infection with retrovirus carrying gene of interest
how can transgenic animals be generated?
some of DNA injected into embryos may be incorporates into the genome
transgenic animals
useful tools for investigating a variety of diseases
how can genes be knocked out?
genes can be disrupted (knocked out) when a foreign fragment is inserted by homologous recombination (genetic information between 2 similar sequences exchanged) - commonly used in DNA repair in cell
how are transgenic mice produced? (4)
- targeting vector used to disrupt sequence of specific gene in mouse embryonic stem (ES) cells
- ES cells containing mutated gene injected into host embryos, then transferred to foster mother - allowed to develop to term
- offspring derived from injection embryos are chimeras, contain cells from black host embryos and brown ES cells
- different coat colours provide useful method of recognising chimeric offspring, then used for crossbreeding to produce fully transgenic mice
CRISPR (2)
- clustered regularly interspaced palindromic sequences
- found in bacterial DNA
genome editing using CRISPR-Cas9 (3)
- Cas9 endonuclease directed to target by guide sequence and cleaves it
- double strand break in target sequence can be repaired by non-homologous end joining, producing random insertions and deletions, inactivating target gene
- more precise change to sequence can be introduced by providing ‘donor’ sequence for homologous recombination (HR)
Cas proteins
near repeated clusters of genes encoding CRISPR-associated (i.e. Cas) proteins, function as “immune system” to bind and cleave foreign DNA sequences
gene silencing by RNA interference (siRNAs)
- double strand RNA (dsRNA) containing strand complementary to specific mRNA can be introduced into a cell
- introduced RNA cleaved into small fragments, small interfering RNAs (siRNAs) by the enzyme dicer
- complementary fragment to mRNA becomes incorporated into RNA-induced silencing complex
- associated complementary RNA guides complex to mRNA which is then degraded
- useful to study gene function
what produces monoclonal antibodies?
immortal cell lines, identical antibodies produced by clones of single antibody producing cell
how are immortal monoclonal antibody producing cells generated? (hybridoma cells)
generated by fusing normal, short-lived antibody-producing cells with immortal cells from a type of cancer called multiple myeloma - results in hybrid cells called hybridoma cells
