Term
| which technique is used for measuring protein levels? |
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Definition
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Term
| northern blotting, RT-PCR and microarray (GeneChip) techniques are all used for measuring what? |
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Definition
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Term
| what are labelled copies of genes amplified by PCR also known as? |
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Definition
probes
many techniques for measuring nucleic acid levels require probes |
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Term
| name the three types of probes that can be used |
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Definition
Radioactive
Chemical
Fluorescent |
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Term
| autoradiography helps to detect which types of probes? |
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Definition
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Term
which of these probes is high energy and which ones are low?
P32 S35 C14 H3 |
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Definition
P32 - high energy, sensitive, emits Beta radiation
S35 C14 H3 lower energies and sensitivity but HIGHER resolution |
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Term
| which kind of probes involve antigen incorporated into DNA? |
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Definition
| chemical probes - these are bound by enzyme linked antibodies and make a coloured or chemiluminescent compound |
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Term
true or false?
there are many different fluorescent compunds availables for probes |
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Definition
| true - you also need specialised equipment to detect them. |
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Term
| labelled nucleotides (probes) incorporated into DNA are done in vitro by which enzyme? |
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Definition
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Term
| why is an alkaline solution not used for Northern blotting? |
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Definition
| because RNA is degraded by alkaline solutions |
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Term
| nitrocelluose is NOT used for northern blotting. why? |
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Definition
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Term
Northern Blotting
mRNA is size fractioned on ........... gel.
... transferred to membrane by blotting
hybridised with ........ probe
detection by c.......l and a.............y |
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Definition
agarose
RNA
labelled
chemical
autoradiography |
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Term
true or false?
northern blotting gives a measure of transcript size and transcript level |
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Definition
True
- distance moved in gel gives measure of transcript size
- intensity of signal gives measure of how many transcripts exist eg. transcript level.
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Term
| if fluorescent molecules absorb light at one wavelength, will the light emitted be of a higher or a lower wavelength? |
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Definition
higher wavelength.
this can be observed using a fluorescent microscope |
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Term
| if you want to extract RNA or synthesis ss cDNA what technique would you use? |
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Definition
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Term
| if you want to carry out PCR using gene-specific primers which technique would you use? |
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Definition
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Term
| what technique seperates products on agarose gel? |
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Definition
gel electrophoresis
extra info: the amount of product is related to the initial transcript number |
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Term
| which technique is simple, quick and cheap and replaced Northern blotting for routine analysis? |
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Definition
RT-PCR
you get more accurate results from real time RT-PCR as it measures the amount of product at each PCR cycle |
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Term
| population of labelled cDNAs are also known as? |
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Definition
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Term
| a set of DNA fragments (cDNAs, oligonucleotides) attatched to a microscope slide is also known as? |
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Definition
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Term
| a collection DNA oligonucleotides on a stamp-sized chip is also known as? |
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Definition
GeneChip
this is manufactured by Affymetrix using photolithography |
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Term
| dna on a the microarray might be cDNA ..... fragments, whole ....... generated by PCR or oligonucleotides corresponding to part of the ORF (open reading frame) |
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Definition
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Term
| what do most microarray experiments compare? |
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Definition
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Term
true or false?
fluorescent molecules absorb and emit light at the same wavelength? |
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Definition
false
they absorb at one wavelength but emit at a second, higher wavelength. |
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Term
| which method can detect a particular protein in a complex mixture? |
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Definition
western blotting
this uses SDS-polyacrylamide gel electrophoresis (PAGE) followed by blotting |
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Term
| what method would you use to measure gene copy number and organisation? |
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Definition
| Southern blotting - allows you to analyse DNA eg, presence/absence of a DNA sequence; measure gene number |
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Term
| what method would you use to measure mRNA size and abundance? |
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Definition
| Northern blotting allows measurement of gene expression and relative transcript abundance |
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Term
| which technique would determine the distribution of a mRNA in a whole organism or tissue? |
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Definition
| in situ hybridization allows location of gene expression in whole organism |
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Term
| which technique measures abundance of a very large number of mRNAs simultaneously? |
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Definition
| Microarrays this allows whole transcript analysis providing absolute and relative levels of gene expression |
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Term
| which technique determines protein size and relative abundance? |
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Definition
| Western Blotting allows detection and and semi-quantification of final protein product |
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