ABT 211 BASIC MOLECULAR TECHNIQUES

BASIC MOLECULAR TECHNIQUES

I. TISSUE STORAGE

1. Cryo-preservation

The ideal storage for molecular specimens is by using cryo-preservation, storage in liquid nitrogen or at -80ºC.

2. Storage in ethanol

Ethanol preserved samples are regularly used in DNA molecular studies. However the quality of the alcohol used can play a large part in the condition of the DNA in a specimen. Preservation in absolute ethanol offers the best means of preserving both the DNA and the gross morphology of a specimen. DNA extracted from specimens preserve in absolute ethanol are of:

• High molecular weight
• High quality

Disadvantages of ethanol preservation:

• Can cause extensive tissue shrinkage
• Colour loss
• Extract cellular components such as lipids

3. Storage in RNA later

This reagent was designed for the storage of tissue samples from which you can extract RNA. However tissue stored in this buffer can be also used to extract DNA successfully of high molecular weight and quality RNA later ® is an aqueous, non toxic, tissue storage reagent that rapidly permeates most tissues to stabilize and protect RNA in fresh specimens. RNA later eliminates the need to immediately process or freeze samples; the specimen can simply be sub- merged in RNA later and stored for analysis at a later date.

Storage and stability

• Store RNA later at room temperature. It is guaranteed for 6 months from the date of receipt, properly stored.
• Samples in RNA later can be stored for extended periods under conditions where RNA degradation would normally take place rapidly. Tissues can be stored indefinitely in RNA later at –20°C or below.
• RNA later can be safely discarded down the sink and flushed with water.

Materials compatible with RNA later

RNA later can be used for RNA preservation with most tissues, cultured cells, bacteria, and yeast. RNA later may not be effective in tissues that are poorly penetrated by the solution, such as waxy plant tissue and bone. RNA later has been extensively tested with animal tissues including, brain, heart, kidney, spleen, liver, testis, skeletal muscle, fat, lung, and thymus. RNA later has also been proven effective for RNA preservation in E. coli, Drosophila, tissue culture cells, white blood cells, and some plant tissues.

4. Whatman FTA Cards

FTA cards are designs for room temperature collection, shipment, archiving and purification of nucleic acids from a wide variety of biological samples for PCR analysis:

• Blood
• Cultured cells
• Buccal cells
• Plant material
• Bacteria
• Plasmids
• Microorganisms
• Solid tissue
• Viral particles

FTA cards are impregnated with a patented solution that lyses cell membranes and denature proteins upon contact. Nucleic acids are immobilized and protected from UV damage and microbial fungal attack. FTA cards are available in several sizes to meet specific needs. Since captured nucleic acids are stabilized, FTA Cards facilitate sample collection in remote locations and simplify sample transport. For example, you can collect samples in the field without worrying about immediate refrigeration. Ship your samples back to the laboratory without expensive special handling or dry ice and process at your convenience.

Store nucleic acids at room temperature for years

Genomic DNA stored on FTA Cards at room temperature for over 17 years (and counting) has been successfully amplified by PCR. RNA, being chemically less stable than DNA, is best analyzed upon return of samples to the laboratory. Frozen storage is helpful for RNA preservation.

Sample integrity is optimized when FTA Cards are stored in a Multi-Barrier Pouch with a Desiccant Packet. FTA Cards offer a compact room-temperature storage system that reduces the need for precious freezer space.

1.1 DNA EXTRACTION

DNA Extraction or rather, nucleic extraction is the process by which nucleic acids are liberated from and then purified away from other cellular materials. There are three basic and followed by selective recovery of nucleic acids from the cellular lysate :

1. Physical Extraction- Breaking the cells open commonly referred to as cell disruption or cell lysis to expose the DNA within. This is commonly achieved by grinding, homogenization, bead beating or sonicating the sample.

2. Chemical dissociation – the solubilisation of lipids (cellular, nuclear, cytoplasmic and organellar membrane systems) using powerful detergent solutions (with appropriate buffer conditions (TRIS, EDTA)) such as:

• SDS (Sodium Docecyl Sulphate)
• CTAB (Cetyl Trimethyl Ammonium Bromide )

For bacterial plasmid preparation:

• SDS and NaOH is used to bust open the cells, followed by neutralisation and precipitation of cell wall and protein with potassium acetate mitochondrial DNA

3. Enzymatic dissociation

DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease. Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenolchloroform and centrifuged the proteins will remain in the organic phase and can be drawn off carefully. The DNA will be found at the interface between the two phases.

4. Recovery of Nucleic Acids

4.1 Organic Extractions to Precipitate Proteins

       phenol -           (phenol + chloroform) –    chloroform

• Organic compounds such as phenol and chloroform are immiscible with aqueous solutions, yet denature and precipitate out proteins from them, whilst leaving nucleic acids untouched
• Different subsets of proteins are precipitated by the different organic compounds. Therefore mixing these compounds with a DNA lysate and then separating the aqueous and organic phases by brief centrifugation (the aqueous phase is the top (lighter) one) clears the aqueous phase of proteins, which either form a pellet at the bottom of the tube or a layer at the interface between the phases.
• Because pure chloroform can be partly miscible with water, it is always used as a 24:1 mix with isoamyl alcohol, which allows for a clearer interface between the organic and aqueous phases. Hence, chloroform refers to this 24:1 mix.
• Extraction can either be undertaken sequentially with phenol, then with a 1:1 mix of phenol-"chloroform", and finally with "chloroform", or 2 rounds of extraction with a 1:1 mix of phenol-"chloroform" can be performed

4.2 Alcohol Precipitation

Recovery of nucleic acids by alcohol precipitation, usually ice-cold ethanol or isopropanol will aggregate together, giving a pellet upon centrifugation. The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to remove the salt previously added.

DNA STORAGE

Extracted DNA should be stored as follows:

• -20ºC or below for long term storage
• DNA for storage should be re-suspended in a buffer solution such as 10 mM Tris HCL pH 7.5 rather than water. If the pH of water is not neutral but slightly acidic this can cause the DNA to degrade upon storage.
• DNA can be stored lyophilized at -20ºC or below for long term storage

1.2 PCR – PRINCIPLES AND PROCEDURES

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

• DNA template that contains the DNA region (target) to be amplified.
• Two primers that are complemtary to the 3’ (three prime) ends of each of the sense and antisense strand of the DNA target.
• Taq polymerase with a temperature optimum at around 70 °C.
• Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
• Buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
• Divalent cations, magnesium or manganese ions ; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis
• Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube

2. PCR-PROCEDURE

Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot start PCR.

• Hot-Start PCR a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.

Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used.

Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'- phosphate group of the dNTPs with the 3’-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.

Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential amplification of the specific DNA fragment.

Final elongation: This single step is occasionally performed at a temperature of 70– 74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining singlestranded DNA is fully extended.

Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

3.0 AGAROSE GEL ELECTROPHORESIS

Agarose electrophoresis is a method used where charged molecules in solution, mainly nucleic acids and proteins, migrate in response to an electrical field. The rate of migration through the electrical field depends on:

• the net charge of the nucleic acid /protein
• the size and shape of the molecules
• the ionic strength, viscosity, and the medium through which the molecules are moving

3.1 Factors affecting migration of nucleic acids

• In an electric field, molecules migrate through the gel towards the anode (Negative-Positive) due to their overall negative charge. Nucleic acids have a natural negative charge due to their sugar-phosphate backbone.
• Separation of molecules on the gel is influenced by the molecule size and the effective pore size of the gel.

·         Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA moves. But voltage is limited by the fact that it heats and ultimately causes the gel to melt

·         Conformations of DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest: nicked or open circular, linearised or supercoiled plasmid)

3.2. Percent agarose and resolution limits

Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus.

The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because they result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with pulse field electrophoresis (PFE).The following is a list of recommended gel percentages for the resolution of nucleic acids in electrophoresis:

3.3 Electrophoresis Buffers

There are a number of buffers used for agarose electrophoresis. The most common being:

·         Tris/Acetate/EDTA (TAE). TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product.

·         Tris/Borate/EDTA (TBE). TBE has a greater buffering capacity and will give sharper resolution than TAE.

3.4. Visualisation of DNA with Gel Red

Gel Red is used to make DNA or RNA bands visible in an agarose gel. It fluoresces under UV light (excitation at 300nm, emission at 595nm) when intercalated into DNA (or RNA)

FEATURES

·         Safer than EtBr (ethidium bromide)

·         Easy disposal - Passed environmental safety tests for direct disposal down the drain or in regular trash

·         Ultra-sensitive - Much more sensitive than EtBr and Safe Sensitivity: Bands of 0.25ng can be detected

·         Extremely stable -Available in water, stable at room temperature for long-term storage and microwavable

·         Simple to use -Very simple procedures for either pre-cast and post gel staining

·         Perfectly compatible with a standard UV transilluminator - Gel Red replaces EtBr with no optical setting change

Dilution of Gel Red:

1. Add 2ul of the concentrated Gel Red stock to 1000 ul of Gel loading Buffer. Mix 2ul of this loading buffer with your sample. OR

2. Add 5 ul of Gel red to 50 ml of agarose solution.

3.5. Gel Loading Buffer

Loading dye is mixed with DNA samples for use in agarose gel electrophoresis. These buffers serve three purposes:

·         They increase the density of the samples, ensuring that the DNA sinks evenly into the well. (glycerol, sucrose or Ficoll)

·         They add colour to the sample, thereby simplifying the loading process.

·         They contain dyes that in an electric field, move toward that anode at predictable rates.

Tracking Dyes

The most common means of monitoring the progress of electrophoretic separations by following the migration of tracking dyes that are incorporated into the loading buffer.

Two widely used dyes displaying different electrophoretic mobilities are bromophenol blue and xylene cyanol.

·         Xylene cyanol typically migrates at approximately 4kb equivalence. So do not use this if you want fragments of 4kb.

·         Bromophenol blue migrates at a rate equivalent to 200-400bp DNA. If you want to see fragments near this size (ie. Anything smaller than 600bp) then use the other dye because bromophenol blue will obscure the visibility of the small fragments.

3.6 Size Markers

There are lots of different kinds of DNA size markers. In the old days the cheapest defined DNA was from bacteriophage so alot of markers are phage DNA cut with restriction enzymes. Many of these are still very popular eg, lambda HindIII, lambda PstI, PhiX174 HaeIII. These give bands with known sizes but the sizes are arbitrary. Choose a marker with good resolution for the fragment size you expect to see in you sample lanes. Companies have started producing ladder markers with bands at defined intervals, eg. 0.5, 1, 1.5, 2, 2.5kb and so on up to 10kb. If you know the total amount of DNA loaded into a marker lane, and you know the sizes of all the bands, you can calculate the amount of DNA in each band visible on the gel. This can be very useful for quantifying the amount of DNA in your sample bands by comparison with the marker bands.

For example HyperLadder I is a typical ready-to-use molecular weight marker.

• Hyper Ladder I produces a pattern of 14 regularly spaced bands, ranging from 200 to 10,000bp. The 1,000 and 10,000bp bands have the highest intensity to allow easy identification. The size of each band is an exact multiple of 100bp.  

• SIZING- when using the standard loading of 5μl per lane, (720ng of DNA) each band corresponds to a precise quantity of DNA (see figure)

                                 

3.7 TROUBLESHOOTING

Common problems encountered in agarose gel electrophoresis are described below, along with several possible causes.

·         Poor resolution of DNA fragments. The most frequent cause of poor DNA resolution is improper choice of agarose concentration. Low percentage agarose gels should be used to re-solve high-molecular-weight DNA fragments and high percentage gels for low-molecular- weight DNAs (see Table 2.5A.1). Fuzzy bands, encountered particularly with small DNA fragments, result from diffusion of the DNA through the gel. This is especially true when gels are run for long periods of time at low voltages.

·         Band smearing. Trailing and smearing of DNA bands is most frequently observed with high molecular-weight DNA fragments. This is often caused by overloading the DNA sample or running gels at high voltages. DNA samples loaded into torn sample wells will also cause extensive smearing, as the DNA will tend to run in the interface between the agarose and the gel support.

·         Melting of the gel. Melting of an agarose gel during an electrophoretic separation is a sign that either the electrophoresis buffer has been omitted in the preparation of the gel or has become exhausted during the course of the run. For high-voltage electrophoresis over long time periods, TBE should be used instead of TAE as it has a greater buffering capacity. Also, minigel and midigel boxes, which typically have small buffer reservoirs, tend to exhaust buffers more readily than larger gel boxes.

4.0 CYCLE SEQUENCING

DNA sequencing is the process of determining the exact order of the bases A, T, C and G in a piece of DNA. In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA.

Cycle sequencing is a modification of the traditional Sanger sequencing method. The components are DNA, primer, heat resistant DNA polymerase, 4 dNTP´s, 4 ddNTP´s (dideoxy terminator nucleotides) fluorescently labelled with four different dyes and buffer containing MG++ and K+. The single primer binds to the complementary DNA strand and is extended in a linear mode. This extension continues until by chance and depending on the complementary base a particular ddNTP is incorporated. Because of the latter’s dideoxy-configuration the polymerase cannot add any other base to this fragment and the extension is terminated. Thus at the end of the selected number of cycles, numerous fragments with different lengths and one labelled nucleotide at the end are generated. Stoichiometric manipulation of the reaction components ensure that the fragments of every possible length starting from n+1 say 2000 bases are generated with n being the number of bases in the primer. The key difference between the traditional Sanger method and cycle sequencing is the employment of a thermo stable DNA polymerase. The advantage of using such a polymerase, is that the sequencing reaction can be repeated over and over again in the same tube by heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers and polymerise new strands. Therefore less template DNA is needed than for conventional sequencing reactions. Also all 4 fluorescnce ddNTPS are in one single reaction, compared to 4 separate reactions for the old isotopic system.

After a post sequencing reaction cleanup, the samples are electro kinetically injected into the array of 96-capillary sequencers. The negatively charged fragments migrate toward the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be read as the fragment’s base sequence. A laser beam excites these dye molecules as the fragments reach a detection window, producing fluorescent signals that are collected from all 96-capillaries at once, spectrally separated and focused onto a CCD (charge coupled device) camera. Sophisticated optical and electronic devices produce a colour readout that is translated with the help of sequence analysis software into a sequence as we see it.