Term
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Definition
| Used density gradient centrifugation strategies to isolate DNA in 1958 to show that DNA replicated semi-conservatively. |
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Term
| Differential Density Gradient Centrifugation for WBCs |
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Definition
| White blood cells (WBCs) are isolated from red blood cells (RBCs) or bone marrow by mixing with isotonic solution overlaid with ficoll (branched sucrose polymer that doesn't penetrate biological membranes). After centrifugation, WBCs settle into a ficoll layer between the plasma and RBCs. The WBCs are removed and washed by resuspension and centrifugation in saline before DNA isolation. |
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Term
| Differential Lysis of WBCs |
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Definition
RBCs and WBCs have different osmotic fragilities. When whole blood or bone marrow is incubated in hypotonic buffer or water, the RBCs will lyse before the WBCs.
WBCs are pelleted by centrifugation, leaving RBC membranes and hemoglobin suspended in solution. |
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Term
| 1 mL Buffy Coat DNA Yield |
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Definition
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Term
| 1 mL Bone Marrow DNA Yield |
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Definition
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Term
| 10^7 Cultured Cells DNA Yield |
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Definition
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Term
| 1 mg Solid Tissue DNA Yield |
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Definition
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Term
| 10 mL Lavage Fluid DNA Yield |
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Definition
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Term
| 10 mg Mitochondrial Tissue |
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Definition
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Term
| 100 mL Plasmid DNA Culture DNA Yield |
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Definition
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Term
| 0.5 mL Bacterial Culture DNA Yield |
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Definition
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Term
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Definition
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Term
| 0.5 mL Serum, Plasma, Cerebrospinal Fluid DNA Yield |
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Definition
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Term
| 0.5 - 1 cm Dried Blood DNA Yield |
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Definition
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Term
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Definition
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Term
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Definition
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Term
| 500 mg Bone, Teeth DNA Yield |
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Definition
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Term
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Definition
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Term
| 10 mm2 Fixed Tissue DNA Yield |
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Definition
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Term
| 1 mg Animal Feces DNA Yield |
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Definition
2 - 100 pg
Cells in feces are subjected to digestion and degradation. |
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Term
| Extraction of DNA from Tissue Samples |
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Definition
| Tissue must be dissociated by grinding, homogenizing, or mincing first. Fixed, embedded tissue can be deparaffinized by soaking in xylene and rehydrated by successive soaks in decreasing amounts of EtOH. Fixed tissue can be used without dewaxing for small DNA fragments. |
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Term
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Definition
| DNA in fixed tissues is broken (cross-linked to varying degrees depending on the fixative). How the tissue was handled before fixing and the length of time tissue was fixed also factor in DNA quality. DNA targets of less than 1,000 bp can be consistently isolated, with longer fragments obtainable after Pro K digestion. |
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Term
| Good Fixatives for DNA Quality |
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Definition
10% buffered formalin - 2-5 kbp
Acetone - 2-5 kbp |
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Term
| Mediocre Fixatives for DNA Quality |
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Definition
Zamboni's - 0.2-2.0 kbp
Clarke's - 0.8-1.0 kbp
Paraformaldehyde - 0.2-5.0 kbp
Metharcan - 0.7-1.5 kbp
Formalin-alcohol acetic acid - 1.0-4.0 kbp |
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Term
| Bad Fixatives for DNA Quality |
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Definition
Carnoy's - 0.7-1.5 kbp
B-5 - <0.1 kbp
Zenker's - 0.7-1.5 kbp
Bovin's - <0.1 kbp |
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Term
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Definition
| A carrier molecule used to recover minimal amounts of DNA (yeast RNA and linear polyacrylamide also used). Isolated from mussels, so may contain DNA (can be treated with DNase). A color indicator may be used to help see small pellets. 10-20 ug of carrier is added before incubation and centrifugation. |
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Term
| Extraction of DNA from Microorganisms |
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Definition
| Bacteria and fungi may have cell walls to break down. Mechanical disruption with grinding or glass beads works, but might damage chromosomal DNA. Enzymes that break down cell walls (lyzozyme or zymolyase) can be used. Treatment with a detergent and base in the presence of Tris EDTA and glucose also breaks cell walls. |
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Term
| DNA Extraction by Boiling |
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Definition
| Samples with tough cell walls can be broken down by boiling in dilute sucrose triton detergent, Tris buffer and EDTA after lysozyme treatment. The DNA from this procedure can be immediately precipitated with alcohol. This DNA is single-stranded and may not work for assays requiring double-stranded DNA (such as restriction enzyme digest). |
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Term
| Organic DNA Precipitation |
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Definition
| DNA in the aqeous phase of extraction solution is precipitated with alcohol (iso or EtOH) and salt (ammonium, potassium, lithium/sodium chloride). The alcohol is added to the upper phase solution in 2:1 or 1:1 ratios and DNA forms a precipitate which is collected by centrifugation. 70% EtOH is added to clean out excess salts. The result is centrifuged and the solution is removed before the DNA is resuspended in Tris, EDTA, or water. |
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Term
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Definition
| Once DNA is extracted from the cell, it needs to be cleaned from proteins, lipids, carbohydrates, and cell debris. Done organically by using a high salt, low pH, phenol/chloroform mixture. This dissolves hydrophobic elements and strips away DNA-associated proteins. RNase can be added, or cetyltrimethylammonium bromide (removes polysaccharides). |
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Term
| Phenol and Chloroform Addition in DNA Extraction |
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Definition
When phenol and chloroform are added, a biphasic emulsion occurs. When centrifuged at the correct pH, a hydrophobic layer of lipids forms on the bottom and a hydrophilic phase containing DNA on top. An amphiphilic phase will form as a white precipitate between the layers.
Phenol is caustic so this method is not preferred. |
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Term
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Definition
| Used as a reagent in DNA extraction. Reagent grade EtOH is denatured and has other components mixed in because 100% EtOH can't be distilled. The usual mix is 90.25% EtOH, 4.75% methanol, and 5% isopropanol. |
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Term
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Definition
Usually undenatured (99% iso, 1% water) when used for DNA extraction.
Less volatile than EtOH and can be used at room temperature to precipitate DNA. Less iso is needed to precipitate DNA. |
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Term
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Definition
| A chelating agent added to DNA fated for long-term storage to protect from DNases preset in the environment. Part of TE buffer (10 mM Tris, 1 mM EDTA). Inhibits restriction digest and PCR. If DNA yield is low and the sample will be used for analysis, rehydration in water is preferred. |
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Term
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Definition
| "Salting-Out." Uses low pH and high salt concentration to precipitate proteins and leave DNA in solution. DNA is then precipitated using isopropanol and centrifugation before rehydration. |
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Term
| Solid-Phase DNA Isolation |
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Definition
| Uses solid matrices (columns or beads) to bind and wash DNA. First the cells are lysed using a specific buffer, and then the lysate is applied to the solid-phase in a high-salt buffer, which promotes DNA binding to the matrix. cRNA is used to prevent DNA from binding permanently to the matrix (no other carrier can be used). The immobilized DNA is washed and then eluted with water, TE, or low salt buffer. |
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Term
| Crude Lysis DNA Extraction |
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Definition
| Some procedures either don't need or can't have extended DNA purification, such as screenings of large numbers of sample (e.g., electrophoresis), DNA isolated from limited material, and DNA from different sources (e.g., fixed tissue). Just simple lysis of the cells in the sample should release DNA usable for amplification. |
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Term
| Chelating Resin Extraction |
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Definition
Chelex - a cation-chelating resin used in DNA extraction. Cells are mixed with 10% chelex resin beads and boiled to lyse. DNA is in the supernatant after cooling and centrifuging.
Used in forensic science but also good for DNA purification of fixed samples. |
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Term
| Proteolytic Lysis of Fixed Material |
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Definition
| Fixed tissue is easily accessible and may be the only source of material. This sections are usually used for extraction ( sectioning is not necessary for needle samples). Fixed tissue can be used without dewaxing. Sometimes areas of interest are identified microscopically and then isolated directly using a scraper in buffer or laser capture. Cells must be washed before lysis. Simple screenings just require lysis in detergents. PCR amplification requires lysis buffer and Pro K. The resulting DNA can then go directly to amplification. |
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Term
| Mitochondrial DNA Extraction by Centrifugation |
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Definition
Cells are ground on ice to homogenzie them and spun at 700-2000xg to pellet out cells, cell debris, and nuclei. The supernatant contains the mitochondria, which are pelleted at 10,000-16,000xg. Lysis with detergent follows and proteinase removes contaminants. Cold ethanol is used for suspension and water to resuspend.
High-ionic strength buffers or beta-mercaptoethanol can be used during the homogenization to protect the mitochondria from dissociation. |
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Term
| Mitochondrial DNA Extraction from Total DNA |
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Definition
| Total DNA can be isolated from the cell. The mitochondrial DNA can be analyzed from the total DNA background using PCR or hybridization. |
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Term
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Definition
| RNF. RNA is easily degraded, especially since RNases are everywhere and can renature even after autoclaving and remain active down to 20 C. The must be removed or inactivated before RNA extraction, so an RNF is necessary. Different disposables must be used in this area, and gloves. Reusable glassware can only be used if it is cleaned and then baked at 400 C for 4-6 hours. |
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Term
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Definition
Several types of RNA exist to be extracted. 80-90% of RNA in cells is ribosomal, consisting of the two subunits, which can be separated via eletrophoresis. 2.5-5% is messenger RNA which shows up as a faint background on gels, behind the rRNA bands.
Transfer RNAs and small nuclear RNAs are also present. |
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Term
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Definition
| Added to water or buffer (but not tris) to permanently inactivate RNases. Converts amines to carbamic acid esters. Cross-links RNases through covalent bonding, making them insoluble. Its effects on amines is why it cant be used with Tris, polystyrene, or polycarbonate. |
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Term
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Definition
Vanandylribonucleoside complexes - bind active sites of RNases.
Macaloid clays - absorb RNases.
Ribonuclease inhibitor proteins - can be added to form stable noncovalent complexes with RNases in solution. This might require reducing conditions, so dithiothreitol might be needed. |
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Term
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Definition
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Term
| 1 mL Buffy Coat RNA Yield |
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Definition
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Term
| 1 mL Bone Marrow RNA Yield |
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Definition
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Term
| 10^7 Cultured Cells RNA Yield |
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Definition
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Term
| 1 mg Solid Tissue RNA Yield |
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Definition
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Term
| 1 mg Buccal Cells RNA Yield |
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Definition
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Term
| 1 mm3 Solid Tissue RNA Yield |
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Definition
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Term
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Definition
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Term
| Dissociating Tissue for RNA Extraction |
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Definition
Reticulocytes in blood are lysed by osmosis or separated by centrifugation. When tissues are dissociated they must be kept frozen in liquid N or immersed in a buffer that inactivates intracellular RNases. The pancreas contains an especailly large amount of RNases.
Bacterial and fungal specimens are done the same way. |
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Term
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Definition
| Cell-free mediums (serum, plasma, culture medium) must be used to get viral DNA via special columns or beads. Total RNA extraction methods can't tell between cellular RNA and microorganism RNA, which is why dell-free mediums are needed. |
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Term
| Cell Lysis for Organic RNA Extraction |
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Definition
| Performed in the presence of a high salt or RNase inhibitor in a detergent/phenol. High salt buffers aren't necessary if guanidine isothiocyanate (GITC) is used, as it is a strong denaturant. Strong reducing agents (2-mercaptoethanol) can also be used. |
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Term
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Definition
| Acid phenol (25):chloroform (24): isoamyl alcohol (1) solution works. Chloroform promotes phase separation and denatures proteins. The alcohol prevents foaming. For RNA, this stage must be acidic, which is achieved with an acidic buffer. DNases might be added. After separation, an aqueous solution containing RNA is removed to a clean tube and RNA precipitation happens with iso or EtOH. Yeast-carrier RNA can be added to promote pelleting. Precipitate is washed in 70% EtOH and resuspended in RNA buffer or water. |
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Term
| Solid-Phase RNA Extraction |
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Definition
Strong acidic buffer conditions must be altered before the lysate goes on the column. EtOH must be added. The lysate is added to the column in a high salt chaotropic buffer and then washed. DNase might be added. Washes and eluant are drawn through column with vacuum or centrifugal force. Eluted RNA is usally enough and pure enough for immediate use.
1 million eukaryotic cells/10-5 mg of tissue will yield 10 ug RNA/ |
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Term
| Proteolytic Lysis of Fixed Material RNA |
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Definition
Reagent sets for RNA Extraction from fixed tissue create lysis and incubation conditions that reverse formaldehyde alteration of RNA and release it from tissue without degrading it further.
Spin columns can remove DNA contamination. |
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Term
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Definition
mRNA is only 2.5-5% of total RNA. Most of it is from highly expressed genes, very little from rare or single-copy mRNA. Columns or beads with ssoligomers of thymine or uracil are used to enhance yield. mRNAs contain a polyA tail that will bind to the oligomers. Residual rRNA is washed off, and warm, low-salt buffer with detergent breaks H-bonds and elutes mRNA.
30-40 ng of mRNA from 1 ug total RNA. |
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Term
| Problems of mRNA Isolation |
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Definition
| PolyA/PolyU binding efficiency is variable. Secondary structure in sample can compete with binding to the target oligomer. mRNAs with short polyA tails might not get caught. AT-rich DNA fragments might bind instead. DNase can't be used on the mRNA before it is bound due to potential digestion of oligo-conjugated matrices. rRNA might purify with mRNA leading to a product not purely mRNA. |
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Term
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Definition
| Fluorescent dyes (ethidium bromide, SyBr Green) which bind to DNA can also detect RNA. Silver stains can also detect small amounts of DNA. |
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Term
| Good Gel Electrophoresis DNA Bands |
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Definition
Plasmid DNA should be a bright, moderatly mobile single band with no other bands (nicked or broken plasmids).
High-molecular weight Chromosomal DNA should be a bright band with low mobility. |
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Term
| Good Gel Electrophoresis RNA Products |
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Definition
| High-quality RNA will result as two bands, one for the 28s rRNA and one more mobile for the 18s rRNA. If these bands are smeared or absent the quality of RNA is unacceptable. |
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Term
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Definition
Concentration can be determined from absorptivity constants (50 for dsDNA, 40 for RNA)
A = ebc
A = Absorbance
e - Molar absorptivity (M/mol-cm)
b - Path length (cm)
c - concentration (mg/L) |
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Term
| Concentration from Absorbance |
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Definition
If A = ebc, A is directly proportional to concentration at that wavelength.
One absorbance unit at 260 nm equals 50 mg/L of dsDNA and 40 mg/L of RNA.
Phenol absorbs UV light at 270-275 nm so residual phenol left from organic extraction can increase 260 readings. |
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Term
| Common Spectrophotometry Contaminants |
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Definition
230 nm - Organic compounds
270 nm - Phenol
280 nm - Protein
>330 nm - Particulate matter |
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Term
| Purity Assessment via Spectrophotometry |
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Definition
| If a concentration is read over a range of wv, contamination can be calculated. The A reading an be graphed as a function of wv, producing a curve that should peak at 260 nm for nucleic acid. However, most solutions are read at 2 or 3 wv to detect contamination at wv other than 260 nm. |
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Term
| Protein Contamination and Spectrophotometry |
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Definition
Nucleic acids are most likely to be contaminated by proteins, which are abundant and linked to DNA via aromatic tryptophan and tyrosine residues. Proteins absorb light at 280 nm.
Nucleic acid should have an A of 1.6-2 times more at 260 nm than 280 nm, so a 260:280 of <1.6 indicates protein contamination.
Purification of the sample may be necessary. pH also affects 260/280 readings, so alkaline buffers are recommended. |
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Term
| RNA Contamination and Spectrophotometry |
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Definition
| RNA has a high 260:280, 2.0-2.3. A 260:280 of over 2.0 might indicate RNA contamination. Some DNA analysis procedures are unaffected by RNA, but others are. RNase can be used to eliminate it. |
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Term
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Definition
| Fluorescent Spectroscopy. Uses DNA-specific dyes to measure fluorescence related to DNA concentration. Won't measure single nucleotides or degraded DNA. |
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Term
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Definition
3,5-diaminobenzoic acid 2 HCl. A dye used for DNA fluorometry. Combines with alpha methylene aldehydes (deoxyribose) to fluoresce.
Used to detect nuclei and as a control in spot integrity and hybridization in microarray analysis. |
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Term
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Definition
Used in DNA fluorometry. Joins A-T pairs in the minor groove of DNA, so is specific to intact dsDNA. DNA with unusually high or low GC content complicates binding. The standard used to measure concentration by fluorometry must have GC content comparable to the sample. Calf thymus DNA (GC = 50%) is used for unknown DNA concentrations.
Hoescht dye is sensitive to 200 ng DNA/mL. |
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Term
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Definition
| DNA-specific dyes used in fluorometry. PicoGreen is sensitive down to 25 pg DNA/mL due to its bright fluorescence when bound to dsDNA. Won't bind to ssDNA or RNA. |
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Term
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Definition
| A DNA-specific dye used in fluorometry. Will bind to oligonucleotides. Will detect down to 100 pg/mL of ssDNA. Won't fluoresce when bound to dsDNA or RNA. |
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Term
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Definition
| Can measure RNA in solution via fluorometry. Intensity is 20-26% lower with polyadenylated RNA than total RNA. Will still bind to DNA, so DNase must be used. Sensitive to 2 ng/mL. |
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Term
| Spectrophotometry vs. Fluorometry |
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Definition
Spectrophotometry - Can detect single nucleotides. Can't distinguish between DNA and RNA. Simple to prep samples. Not accurate at low concentrations.
Fluorometry - Won't detect single nucleotides. Can distinguish DNA and RNA. Samples need dye mixing prep. Accurate at low concentrations. |
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