Term
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Definition
Under specific conditions, restriction enzymes have a high fidelity for the sites they cut at. If conditions are not correct, they might cut at DNA sequences not normally targeted. The degree of nonconforming activity under improper conditions varies with the enzyme.
Conditions causing star activity: contamination, suboptimal buffer, lengthy reaction time, high enzyme concentration, and divalent cation imbalance. |
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Term
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Definition
A matrix (usually nitrocellulose) with high affinity for proteins and nucleic acids. DNA fragments from restriction enzyme digests that have been separated on a gel can be denatured and transferred to the matrix. A labeled probe is added to the blot and will bind with complimentary regions of interest. This identifies specific sequences and their presence or absence. Any gene can be analyzed at a molecular level as long as the sequence is known.
Used for analyzing gene structure. |
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Term
| Southern Blot Restriction Enzyme Digest |
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Definition
| Test DNA is digested with multiple restriction enzymes. Each restriction enzyme digest requires 10-50 ug of DNA (depends on the sensitivity of the assay and the abundance of the target). Restriction enzymes are diluted to 1/10 with buffer. Each sample has a separate digestion for each restriction enzyme. Digestion is 1-3 hours long to ensure all DNA is cut. Highly specific restriction enzymes are used. |
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Term
| Southern Blot Gel Electrophoresis |
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Definition
| Restriction digest fragments are resolved on gels. Composition of gel and run time depend on fragment length (16 hours and 20 A for 1,000 length). The resulting gel stain should show long smears for each digest, reflecting the many different sizes of fragments. The brightness of the smears shouldn't vary much from lane to lane. |
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Term
| Faulty Southern Blot Gel Electrophoresis Results |
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Definition
DNA clumped at the top of the lane - Restriction enzyme activity not complete.
DNA clumped at the bottom of the lane - degraded DNA.
Restriction digests should be repeated if there is any uncut DNA. Re-isolation and purification may be required in the case of degraded DNA. |
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Term
| Southern Blot DNA Denaturation |
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Definition
Before gel resolved fragments can be blotted, they must be single-stranded so that the marker can anneal to them. Denaturation happens on the gel. Short fragments are denatured by NaOH, which breaks H-bonds.
Longer fragments may also need to be depurinated by soaking in HCl, which removes purines from the DNA backbone and makes H bonds easier to break. |
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Term
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Definition
| Bind to both proteins and nucleic acids. A versatile medium that can be used with different transfer buffers and detection systems. Isn't sturdy, and become brittle with repeated use. Reinforced membranes can be used for procedures that require multiple probings. |
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Term
| DNA Binding to Southern Blot Membranes |
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Definition
Membranes have a positive charge, so negatively charged ssDNA molecules will bind to them with strong noncovalent bonds in a hydrophobic and electrostatic interaction.
70-150 ug of nucleic acid can bind per cm2 of a nitrocellulose membrane. DNA fragments of 300-20,000 b can fit through membrane pores of 0.05-0.45 um.
Bound DNA strands can still form H-bonds with complimentary sequences (probes). The bond between the DNA and the membrane is stronger, though. |
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Term
| Mechanically Stable Membranes |
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Definition
| These membranes can be made to have a net neutral charge to increase the specificity of binding. Binding capacity can be up to 400 ug/cm2, allowing for high test sensitivity. Once DNA is on the membrane it is exposed to UV light to cause cross-linking. |
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Term
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Definition
Diethlaminoethyl (DEAE)-conjugated cellulose membranes can be used for DNA or proteins.
Polyvinylidenedifluoride (PVDF) and charged carboxyl methyl cellulose membranse are used only for Western blots. PVDF has a binding capacity of 20-40 ug/cm2 - 150ug/cm2 for proteins via hydrophobic and ionic interactions.
Modifications for different methods include small-pore-size membranes for small proteins and membranes for fluorescent detection. |
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Term
| Gel to Southern Blot Transfer |
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Definition
| Before they can be exposed to probes, denatured and electrophoretically separated DNA fragments must be transferred to a solid that will facilitate probe binding and detetion. All the DNA must be moved from the gel to the substrate. The membrane must be thoroughly soaked in buffer before the transfer, dry spots will prevent DNA binding. Membranes should be handled with care, no folding or creasing. |
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Term
| Southern Blot Capillary Transfer |
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Definition
| A reservoir filled with high salt buffer (10x saline sodium citrate) is filled with filter paper. The gel is placed on top of the papers, with the nitrocellulose membrane right on top of it. Dry absorbent paper is placed atop the membrane. Via capillary action the buffer moves through the gel and the membrane into the dry paper. This carries the DNA fragments out of the gel into the membrane, where they bind. Simple, but prone to bubble formation and takes a long time (hours). |
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Term
| Southern Blot Electrophoretic Transfer |
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Definition
An anode is placed above the membrane and a cathode is placed below the gel. The electric current carries DNA fragments from the gel to the membrane.
Can be done in a "tank" with the current transferred via buffer, or in a "semidry" way where the electrodes sit on the gel-membrane sandwich directly and buffer is just used to soak the gel and membrane.
Tank method is good for large proteins and the semidry for small proteins.
Faster than capillary transfer. |
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Term
| Southern Blot Vacuum Transfer |
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Definition
The gel and membrane are placed in circulating buffer and vacuum suction is used to move DNA from the gel to the membrane. This prevents air from getting trapped between the gel and the membrane and causing discontinuous DNA transfer.
Faster but more expensive than capillary transfer. |
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Term
| Southern Blot DNA Binding |
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Definition
| To covalently attach the denatured DNA fragments to the membrane, the membrane can be baked at 80 C for 30-60 min or lit by UV light. This cross-links the DNA to the membrane, keeping the DNA fragments immobilized even when the membrane is washed and re-probed. |
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Term
| Southern Blot Prehybridization |
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Definition
After DNA is attached to the membrane, the membrane is soaked in the same buffer that hybridization occurs in (sans probe), at the same temperature. Salmon sperm DNA or dernhardt solution might be used in buffers for DNA, or formamide for RNA procedures. The soak lasts for over 30 min.
Prevents the probe from attaching to nonspecific sites on the membrane and causing background noise. |
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Term
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Definition
| Modification of Southern Blots used for RNA analysis. Used to examine gene expression, RNA abnormalities (alternative splicing). |
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Term
| Northern Blot Gel Electrophoresis |
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Definition
| 038-1.5 concentration agarose gels. RNA must be at least 30 ug of total RNA or 0.5-5.0 ug polyA RNA. Denaturing conditions must be used during electrophoresis for accurate results. This makes a denaturing step later on unnecessary. |
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Term
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Definition
Gel must be soaked in deionized water to remove the denaturing agent (which will prevent RNA from binding to the cellulose).
Transfer happens in 10/20x SSC or 10x SSPE to a nitrocellulose membrane. |
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Term
| Northern Blot Hybridization |
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Definition
If glyoxal is used to denature RNA, the gel must be soaked at 65 C in tris to remove the denaturant before prehybridization.
Prehybridization and hybridization are the same as for Southern blots. |
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Term
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Definition
| Modification of Southern blot used for proteins. Proteins can be separated via polyacrylamide gel, IEF gels, or dithiothreitol or 2-mercaptoethanol (separate proteins into subunits). |
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Term
| Western Blot Gel Electrophoresis |
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Definition
5-20% polyacrylamide gel. 1-50 ug of protein needed. A denaturant is added to the samples before loading. The most accurate separation can be gotten from running IEF gels and then using polyacrylamide gels, or via two-dimensional gel electrophoresis. Protein standards are run at the same time.
Denaturing gels may affect epitopic sites on proteins from binding to antibodies. Mild buffers (20% glycerol, 50 mM tris-HCl)can be applied to the gel to renature proteins. |
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Term
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Definition
| ssNucleic acid fragments (DNA or RNA) with a signal-producing moiety. The probe needs to hybridize specifically to the sequence of interest, since there is much nucleic acid present. Modified nucleic acids like peptide or locked nucleic acids with normal bases but modified backbones can be used. These resist nucleases and hybridize better to DNA/RNA due to the low negative charge of the backbone. |
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Term
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Definition
| Specific binding proteins or antibodies. Labeled secondary antibodies are then applied to visualize the protein of interest. |
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Term
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Definition
Originally created by cloning the gene of interest and putting it in a plasmid to be cut by restriction enzymes. The probe can then be denatured and labeled.
Sequences can also be isolated from viral genomes or synthesized in the lab. Lab synthesis is only done for short probes. PCR can be used to create many fragments. |
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Term
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Definition
Probe length determines specificity. Can range from 10-10,000 b. Longer probes are more specific.
Short probes are not as specific and can generate a lot of background noise. Not used for Southern blots except for mutation analyses to identify mismatches of one bp. |
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Term
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Definition
Can be more sensitive than DNA probes because RNA:DNA helixes are stronger. Less stable and can't be stored for long.
Coding or complimentary RNA can be used for Southern blots.
Northern blots require complimentary RNA. |
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Term
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Definition
Synthesized from plasmids or PCR templates. cDNA is cloned into a plasmid, linearized, and transcribed. The transcript can be used for Southern blots. Northern blots require the template to be reversed with the primer at the end of the gene to create an anti-sense transcript.
Labeling happens during the in vitro process. RNase can be added after hybridization to reduce background noise, DNase is used to discard the template. Probes are denatured before use. |
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Term
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Definition
Probes used for Western blots. A general response to a specific antigen. They have the ability to bind to multiple epitopes on an antigen, as they are made of a mix of immunoglobins.
To make them, small molecules called haptens can be used to generate an immune response. Immunoglobins can be isolated from the sera by affinity chromatography. |
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Term
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Definition
| Probes used in Western blots. Difficult to produce. More specific as probes, giving less background noise. They only bind to one epitope, so if that is lost the signal is lost. |
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Term
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Definition
Probes need to be labeled and generate a signal. Radioactive 32P has been used, but that's hazardous.
Non-radioactive probes such as biotin and digoxygenin attach to a nucleotide triphosphate (uTP or cTP) which can be attached to the nucleic acid probe. |
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Term
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Definition
| Terminal transferase or T4 polynucleotide kinase adds labeled nucleotides to the end of nucleic acid probes. |
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Term
| Probe Nick Translation Labeling |
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Definition
| DNA polymerase attaches to a nick and displaces the hybridized strand, replacing with labeled nucleotides to the template strand. |
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Term
| Random Priming Probe Labeling |
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Definition
| The probe is denatured to a single strand, and primers of random sequences 6-10 b long will compliment some part of the probe strand and add labeled nucleotides to create copies of the probe strand. |
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Term
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Definition
| Transcribed from cloned/amplified DNA using radioactive, biotinylated, or digoxygenin-tagged nucleotides.These probes are ss, containing only the sequence complimentary to the target. |
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Term
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Definition
| Radioactive 35S can be used, or probes can be attached to enzymes like horseradish peroxidase or alkaline phosphatase. These enzymes produce detectable signals. The labeled antibody might be attached to an unlabeled antibody that binds to the protein of interest by targeting the FC region (two identical protein fragments in antibodies). |
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Term
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Definition
| Probes with internal complimentary sequences may end up folding and with inefficient binding, especially if the folded regions are GC rich. |
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Term
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Definition
Conditions where the probe is exposed to the target. High stringency conditions are particular in probe/target complementarity and length. Overly stringent conditions can lead to no probe binding.
Low stringency conditions don't require as much specificity, but if they are too low probes will bind to unwanted targets, interfering with analysis. |
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Term
| Factors Affecting Hybridization Stringency |
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Definition
- Hybridization temperature (low stringency = low temp, high stringency = high temp)
- Salt concentration of buffer
- Concentration of denaturant
- Length of probe (longer = more stringent)
- Probe sequence (GC rich sequences = more stringency) |
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Term
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Definition
The temperature where half of the nucleic acid probes are ds and half are ss. Expresses amount of energy required to denature that sequence. Used to estimate stringency conditions.
Tm = 81.5 C + 16.6 logM + 0.51(%GC) - 0.61(%formamide) - (600/n)
M = sodium concentration
n = # of bp in probe |
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Term
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Definition
- Hybrid makeup. RNA raises the temperature (RNA:RNA are most stable, DNA:DNA the least)
- Inclusion of modified nucleotides in the probe raises the temperature.
- Complementarity of the probe and the target. Tm decreases by 1.5 C for every 1% difference in sequence. |
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Term
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Definition
For short probes of 14-20 bp.
Tm = (4C x #GC pairs) + (2 C x #AT pairs) |
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Term
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Definition
Sequence complexity, determined by the length of non-repetitive DNA sequences. Complex sequences take more time to reanneal.
C0 - initial DNA concentration
T = time required to reanneal
C0T1/2 = time for half of the ds sequences to reanneal at a specific temperature |
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Term
| Probe-Target Hybridization |
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Definition
Temperatures 25 C below probe Tm are optimal.
Performed in hybridization bags or glass cylinders. The probe is the limiting reagent, so hybridization solution should be low (10mL/100 cm2 of membrane).
Increasing probe concentration will increase sensitivity, to a point.
Denaturants in the buffer lower the required temperature.
Short probes need 1-2 hours to hybridize, long probes need longer (16 hours for 1,000 b probes). Increasing probe concentration or adding inert polymers can speed hybridization up. |
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Term
| Radioactive Probe Detection |
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Definition
After radioactive probes have hybridized to the target sequences, the membrane is washed to get rid of excess probe. Light-sensitive film is applied to the membrane to detect fragments attached to the probe.
Wash conditions are more stringent than hybridization conditions. |
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Term
| Nonradioactive Probe Detection |
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Definition
| Probes with digoxygenin/biotin hybridize to target fragments and unbound probes are washed off. AP conjugate (antidigoxygenin antibody/streptavidin attached to alkaline phosphatase) is applied to the membrane to bind with the probes. Unbound conjugate is washed off and a solution containing a substrate dephosphorylated by AP that produces a signal (light or colors) is added. |
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Term
| Chemiluminescent Probe Detection |
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Definition
For non-radioactive probes bound to alkaline phosphatase. Substrates phosphorylated by AP: dioxetane, acridium ester, isoluminol, ruthenium trisbipyridy.
The AP acts on the substrate in the dark and releases light at the location of the probe. Can be stronger and faster than radioactive detection, but can cuase non-specific results and is hard to time correctly. |
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Term
| Chromogenic Probe Detection |
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Definition
AP-conjugate bound probes can act on substrates such as tetrazolium dye derivatives or nitroblue tetrazolium (NBT) and bromo-4-chloro-3-indoltl phosphate mixes. The BCIP is dephosphorylated by AP and then oxidized by NBT to make a dark blue dye that produces a signal on the membrane.
These reactions are easy to time since you can see the signal develop. Not as sensitive, especially if randomly primed probes are used. |
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Term
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Definition
Can be used to compare restriction digest patterns for known genes to unknown samples. Small deletions of insertions will not be detected unless they affect a restriction site.
Cross-hybridization can occur, creating bands of the same size in all lanes. |
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Term
| Northern/Western Blot Analysis |
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Definition
| Usually used for gene expression, transcript size/processing, protein modification. An internal standard is included in all samples to correct for uneven sample loading or isolation. A positive control transcript is included in lane one. Its thickness shows the expected amount of gene expression. Sample bands may be lighter/thinner (less expression) or thicker. The appearance of the internal control in a lane suggests that the procedure was a success and light bands are accurate depictions of gene expression. |
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Term
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Definition
A type of membrane hybridization for tests that don't need fragment length to be known (expression, mutation, amplification, deletion). Samples are loaded directly to the membrane as dots or slots. These analyses work best with simple products (PCR products, mRNA preparations). Probe hybridization mustb e optimized because cross-linking is undistinguishable from true results. Negative controls consist of fragemtns of equal complexity without the target sequence. Normalization controls are included to correct for loading or sample differences.
Can analyze more samples than a Southern or Western blot. |
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Term
| Limitations of Dot/Slot Blot Assays |
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Definition
| For dot and slot blots, the area of the membranes and the volume of buffer needed to provide enough probe for an adequate signal to be produced limit their size and use. Also, although hundreds of samples can be tested at once on one blot, they can only be tested for one gene or gene product. |
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Term
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Definition
| Probes for many different sequences are immobilized on a membrane and labeled samples are washed over it to attach to the stationary probes. Because the locations and sequences of all the probes are known, these blots can be used to determine the presence of a sequence in a sample and also its signal intensity. |
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Term
| SSOP Reverse Configuration |
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Definition
| Primers in PCR are attached to biotin or digoxygenin at the 5' end, so amplicons contain those molecules. Multiple alleles attach to the membrane, so the patient sample is applied to see where the amplicons bind. |
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Term
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Definition
Massive reverse dot blots consisting of 1,000s of probe targets on a membrane. Radioactive/luminescent signals are used to detect hybridization of samples to target probes. The result can be read by eye or with phosphor tech. Results are compared to controls on an identical membrane.
Area of membrane and amount of sample limit the use of macroarrays. Much sample is needed for so many probes, which can be a problem for limited clinical samples. |
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Term
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Definition
Use treated glass slides (chips) instead of membranes. Spotting tech can deposit up to 80,000 spots on a chip (the human genome has 30,000 genes, so you can spot genes in triplicate).
The targets spotted on the array are usually DNA (cDNA, PCR products, oligomers), but can be RNA or proteins. They are spotted in triplicate and spaced to avoid geographical errors. Samples are usually cDNA but can be genomic DNA, RNA, or proteins. |
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Term
| High-Density Oligonucleotide Arrays |
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Definition
DNA targets are synthesized directly on the chip. DNA is masked, activated, and nucleotides are attached covalently at certain positions on the chip. Labelled samples are applied to the array for probing. 100,000 targets can be applied to the chip.
Used for mutation analysis SNP analysis, and sequencing. |
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Term
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Definition
| Used to assess levels of transcription (gene expression) by comparing to a normal control. The attached probes on the array are exposed to mRNA from treated cells or different cell types. |
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Term
| Comparative Genome Hybridization |
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Definition
| Array CGH. Genomic DNA is isolated, fragmented, and labelled before being hybridized to the chip. This allows for screening of the genome or specific loci for deletions/amplifications. Provides high resolution and defined genetic info, but is limited by the loci attached to the chip. |
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Term
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Definition
| Fluorescent readers pick up the signal and software analyses it. The software corrects for background noise and normalizes it with standards. Signal intensity is averaged from duplicate samples. The results are relative to control values. A variation of 2-3 STD from the mean is significant. |
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Term
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Definition
- Lack of established controls across labs. Not enough data to determine what degree of non-specific binding and cross-hybridization.
- Background noise
- Different sequences have different binding efficiencies under the same conditions (unless designed to all have the ssame Tm)
- Length of probes are limited by requirements for temperature and buffer |
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Term
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Definition
Probes are attached to beads and suspended in liquid, possibly in a 96 well plate to analyze multiple specimens at once.
The beads are color-coded with one fluorescent dye and the sample with another so that presence of a target can be detected. |
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