Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
15 Cards in this Set
- Front
- Back
|
Nucleic Acid Probes |
- ssDNA or RNA - Sequence is complementary to gene of interest - Typically 100-1000bp - Labelled to allow detection |
|
|
Techniques that use probes |
- In situ hybridisation - Dot blots - Colony screening - DNA profiling - Southern blots - Northern blots - Diagnostic tests |
|
|
Nucleic acid hybridisation (Definition, Factors that affect it) |
Nucleic acid hybridisation: probe sequence binds to the sequence of interest via hydrogen bonds Factors to consider: - Probe length - Base composition (affects H bonding) - Chemical environment (salts, detergents) - Temperature (too high = H bonds will break in specimen and probe) |
|
|
Nucleic acid hybridisation (Steps required prior to) |
- Isolation of sample from cells - Quality check of sample (RNA must be high qualityand not degraded) - Treatment of sample (e.g. digestion by RE for southern blot) - Separation of the sample (gel electrophoresis) - Immobilisation of the nucleic acids (immobilised on a solid support, so they do not moveduring hybridisation and washing) |
|
|
Nucleic acid hybridisation (Pre-hybridisation buffers) |
1. Blocking agents high MW polymers and detergents that prevent non-specific binding of probes eg. detergents, bovine serum albumin 2. Rate enhancers eg. dextran sulphate 3. Denaturants eg. urea, formamide (denatures DNA and keeps RNA single stranded. Increased [formamide] = decreased Tm) |
|
|
Nucleic acid hybridisation (Membrane types) |
1. Nitrocellulose: requires blocking agents for both pre-hyb and hyb to stop non-specific binding 2. Nylon: does not require blocking agents in hybridisation step. less problems with non-specific binding |
|
|
Nucleic acid hybridisation (Stringency) |
Stringency: the specificity withwhich a particular target sequence is detected byhybridization to a probe High Stringency only sequences that are 100% complementary will hybridise use HIGH temperature, low salt Low Stringency partially matched sequences will hybridise too use LOW temperature, high salt |
|
|
Nucleic acid hybridisation (Procedure) |
- Wet membrane in 6x SSC (salt, sodium citrate) - Place in hyb bottle with prehyb (0.1mL per cm2);incubate ~ 65°C for 1-2hrs - Pour off prehyb; add fresh hyb solution containingboiled probe;incubate overnight - Pour off probe; wash membrane in 2xSSC with0.5%SDS at RT for 5mins - Wash membrane in 2x SSC with 0.1% SDS for15mins at RT - Wash membrane in 0.1x SSC with 0.1% SDS for0.5 to 4 hrs at 65°C; - Probe can now be detected - Membrane can be stripped and re-probed to detect a different sequence |
|
|
Blotting |
Blotting: immobilisation of nucleic acids onto a solid membrane Northern = RNA Southern = DNA Western = Proteins |
|
|
Southern Blotting |
Determines - if sequence exists in the samples - how much sequence is present Steps - Isolation and purification of DNA - Restriction digests of DNA - Electrophoresis of prepared DNA fragments - Transfer of the DNA from the gel onto a membrane using capillaryaction - Baking or UV cross-link nucleic acid onto membranes - Probing of the membrane with the probe - Detection of the probe |
|
|
Northern Blotting |
Determines - gene expression by RNA analysis - if a gene is being transcribed at different levels in different samples/individuals Steps - Isolation of RNA from samples - Electrophoresis RNA in presence of formaldehyde (denaturing) - Transfer of the RNA from the gel onto a membrane using capillaryaction - Baking or UV cross-link nucleic acid onto membranes - Probing of the membrane with the probe (hybridisation) - Detection of the probe |
|
|
Probe labelling (Types of labels) |
Radioactive - 32P, 33P, 3H Non-radioactive - Biotin - Digoxygenin - Flourescein |
|
|
Probe labelling (Methods of labelling) |
1. Random Priming - Uses Klenow polymerase - Uses linear template - heated to denature dsDNA into ssDNA - Random hexamers asprimers - Klenow polymerase anddNTPs added(One dNTP must be labelled) * Better to use a purifiedsegment of DNA astemplate rather than aplasmid to reducebackground 2. Nick translation - Treat with DNase I to introduce nicks - DNA Pol I catalyses the polymerisation ofnucleotides into dsDNA in a 5´ to 3´ direction - 5´ to 3´ exonuclease activity enables enzyme toreplace nucleotides in the growing strand of * One of dNTPs added to repair nicked DNA is labelled 3. End labelling - Using TerminalDeoxynucleotidyl Transferase which adds dNTPs to the 3´end of DNA - Most efficient on DNA with a protruding 3´ end, less efficient if DNA has a blunt or recessed 3´ end * One of dNTPs added must be labelled 4. End filling - Uses Klenow polymerase - Uses template with sticky ends - Klenow polymerase and dNTPs added * One dNTP must be labelled |
|
|
Detection of Probes |
Radioactive: autoradiography - photographic emulsion contains silver halide crystals - B particles emitted by radioactive decay converts silver ions to silver atoms - observed on X-ray film at -70°C to extend t1/2 Non-radioactive 1. Biotin - binds well to streptavidin - streptavidin can be conjugated to a reporter 2. Digoxygenin or Flourescein - detected with antibodies |
|
|
Applications of probing techniques |
1. Dot blots - dot sample on paper then apply probe to see if + or - results - good for high # tests in short time (eg. HIV) 2. Colony hybridisation - blot colonies to membrane - degrade cells, purify DNA - probe with labelled DNA - autoradigraphy 3. DNA fingerprinting - single probe that identifies length of repeated elements in non-coding DNA 4. FISH - flourescent in-situ hybridisation - use different colours for different target at once - to look at location of target sequences/genes - eg. to ID organism of tissue sample |
