Alkaline Lysis, SDS Page, Sequencing, Chromatography, Agarose Gel Electrophrosis, Probes

Alkaline Lysis

It is method of choice for isolating circular plasmid DNA or even RNA from cells. It is probably one of the most generally useful techniques as it is fast, reliable and relatively clean way to obtain DNA from cells.

Alkaline lysis depends on a unique property of plasmid DNA. It is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the chromosome.

The cell debris is precipited using SDS and potassium acetate. This is spun down, and the pellet is removed. Isopropanol is then used to precipitate the DNA from the supernatant, the supernatant removed, and the DNA is resuspended in buffer.

Procedure
1.Spin down cells : DNA is in the cells, so it is in the pellet. 2. Discard the supernatant : pieces of cell wall released are floating around in the supernatant. These cell wall pieces can inhibit enzyme action on the final DNA, so it is impt to get rid of all the supernatant and to even invert the tube and wipe the lip with a Q-tip.

3. Resuspend the cells: in the buffer i.e. EDTA. EDTA chelates divalent metals (primarily magnesium and calcium ). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells.

4 . Lyse the cells :

This is done with sodium hydroxide and SDS. This highly alkaline solution gave rise to the name to the technique. Mix this by gentle inversion and incubate on ice for five mints ( but no longer, or DNA will be irreversibly denaturized )


a. b.

c.

Three things happen during this stage SDS pops holes in the cell membranes. NaoH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA. NaoH denatures the DNA. Cellular DNA becomes lineraized and the strands are separated. Plasmid DNA is circular and remains topologically constrained.

5.Renature the plasmid and get rid of the garbage

1.

2. 3.

Add potassium acetate (KAc), which does three things Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single stranded DNA (ssDNA). The ssDNA is precipitated, since large ssDNA molecules are insoluble in high salt. Adding potassium acetate to the SDS forms KDS, which is insoluble. This will allow for the easy removal of the SDS from plasmid DNA.

Now that the contaminants can be easily separated and centrifuge to remove the cell debris, KDS and cellular ssDNA. The plasmid DNA is in the supernatant, while all of the garbage is in the pellet.

6. Precipitate the Plasmid DNA by Alcohol Precipitation

Ethanol or isopropanol and a salt such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate and spin this down. DNA is negatively charged, so adding a salt masks the charges and allows DNA to precipitate. This will place DNA in the pellet.

Rinse the pellet plasmid DNA in ice cold 70% EtOH and air dry for about 10 mints to allow the EtOH to evaporate. Resuspend the clean DNA- pellet in buffer EDTA plus RNases to cleave any remaining RNA. The DNA is back in solution.

Uses
DNA of this purity is use for the following. 1. In vitro transcripiton or translation with some enzymes.

SDS Page

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. It is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their functional length of polypeptide chain in or molecular weight as well as higher order protein folding ( in other words according to their electrophoretic mobility )

The SDS portion is a detergent used in shampoo, soap, or toothpaste. The purpose of SDS is to take the protein from the native shape, which is a big glob, and open it up into linear piece. This will allow it to run more efficiently down the gel and will be easier to compare. SDS is an anionic detergent that binds quantitatively to proteins, giving them linearity and uniform charge, so that they can be separated solely on the basis of their size.

Procedure

The solution of proteins to be analyzed is first mixed with SDS, which denatures secondary and non-disulfide-linked tertiary structures, and applies a negative charge to the proteins in proportion to its mass. Adding SDS linearizes the proteins so that they may be separated strictly by molecular wt. The SDS binds to the proteins in a ratio of app 1.1 2.2 g SDS/g protein. This gives a uniform mass: charge ration for most the proteins, so that the distance of migration through the gel is related to the size of the protein.

Electrophoresis and Staining

The denatured proteins are applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer. An electric current is applied across the gel, causing the -vely charged proteins to migrate across the gel. Depending on their size, each protein will move differently through the gel matrix, short proteins will more easily fit through the pores in the gel, while the larger ones will have more difficulty.

After a set amount of time the proteins will have differentially migrated based on their size, smaller proteins will have traveled further down, while the larger ones will have remained closer to the point of origin. Once this is done the gel is fixed so that the proteins in the gel dont come out when the gel is stained. The fixative used is acetic acid 25 % in water. The gel is stained with coomasie blue dye R250 or silver stain, allowing visualization of the proteins .

After staining, different proteins will appear as distinct bands within the gel. It is common to run marker proteins of known molecular wt in a separate lane in the gel, in order to calibrate the gel and determine the wt of unknown proteins by comparing the distance traveled relative to the marker.

Reducing SDS-PAGE

Besides the addition of SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol or 2-mercaptoethanol, which further denatures the proteins by reducing disulfide linkages, thus overcoming forms of tertiary protein folding, and breaking up quaternary protein structure.

Uses

Establishing protein size Protein identification Determining sample purity Identifying disulfide bonds Quantifying proteins Blotting applications

Sequencing

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. The most frequently used methods is the one developed by Frederick Sanger using Chain termination.

p-p-p-OCH2 H H H

Bas H e H H

p-p-p-OCH2 H H OH

o
H H

Base H

Dideoxynucleotide (ddNTP)

Deoxynucleotide (dNTP)

Dideoxynucleotides (ddNTPs) are missing a hyroxyl (OH) group at the 3 position. This position is normally where one nucleotide attaches to form a chain.

Manual Sequencing

To begin the process firstly synthesizing a chain that is complimentary to the template to be analyzed should be done. Add a primer that will anneal. Usually a oligonucleotide primer which is complementary to the template at that region is used.

Divide the sample among four test tubes. One test tube will be used for each specific nucleotide (dGTP,dATP,dCTP,and dTTP). Add DNA polymerase to each tube and one specific nucleotide per tube. Add ddNTPs to all four tubes. Once this is added the chain will not elongate and there is chain termination.

Run this on a polyacrylamide gel using one lane per reaction tube (dGTP,dATP,dCTP and dTTP ). To sequence, read the order of bases from the smallest to the largest.

ddGTP

ddATP

ddCTP

ddTTP

Add ddGTP, ddATP, ddCTP,ddTTP, one to each of four tubes containing target DNA. Load each onto a separate lane on a gel.

Largest

To sequence, read the order of bases from the smallest to the largest. TCGAAGACGTATC

Smallest

Automated Sequencing

An automated sequencer runs on the same principle as the Sanger method. A laser constantly scans the bottom of the gel, detecting bands as they move down the gel. Where the manual method uses a radioactive labeling, automated sequencing uses fluorescent tags on the ddNTPs ( a different dye used for each nucleotide ). This makes it possible for all four reactions to be run in one lane, so that huge numbers of reactions are on the gel.

Dye Terminator Sequencing

An alternative to the labeling of the primer is to label the terminators instead. The major advantage is that complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach.

454 Sequencing

This sequencing was developed in early 2000s. In this method single stranded DNA is annealed to beads and amplified via PCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over the chip, light is produced as ATP is generated when nucleotides are washed over the chip join with their complementary base pairs. Addition of one nucleotide results in a reaction that generates a light signal that is recorded by the CCD camera. The signal strength is proportional to the number of the nucleotides.

RNA Sequencing

RNA is less stable in the cell, and also more prone to nuclease attack. As RNA is generated by transcription from DNA, the information is already present in the cells DNA. RNA molecules are not necessarily co-linear with their DNA template as introns are excised. To sequence RNA, the usual method is first to reverse transcribe the sample to generate DNA fragments. This is then sequenced the above described manner.

Agarose Gel Electrophoresis

This is a method used in biochemistry and molecular biology to separate DNA, RNA or protein molecules by size. This is achieved by moving -vely charged nucleic acid molecules through an agarose matrix with an electric field. Shorter molecules move faster and migrate further than larger ones.

Materials
1. 2. 3. 4. 5. 6. 7. 8. 9.

10.
11.

For AGE several items are needed DNA fragments to separate DNA size markers Buffer solution Agarose Ethidium bromide Nitrile rubber gloves A color marker dye ( bromophenol blue ) and glycerol A gel rack A comb Power supply UV lamp or UV light box

Preparation

Make a 1% agarose solution in 0.5xTBE. Bring the solution to boil to dissolve the agarose in a microwave. Cool the solution to about 600 c at room temperature. Stir the solution while cooling.

Add 1ul ethidium bromide stock per 10 ml gel solution. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack. Insert the comb at one side of the gel about 510mm from end of the gel. When the gel has cooled down and become solid remove the comb. The holes that remain in the gel are the wells or slots. Put the gel, together with the rack, into a tank with 0.5xTBE. Ethidium bromide of the same conc can be added to be buffer. Make sure the gel is completely covered with TBE, and that the slots are at the end electrode that will have the negative current.

Procedure

1.

2.
3. 4.

5.

6.

The agarose gel with three slots Injection of DNA ladder into first slot DNA ladder injected. Injection samples into second and third slot. A current is applied. The DNA moves towards the positive anode due to the negative charges on its phosphate back bone. Small DNA strands move fast, large DNA strands move slowly through gel. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular wt and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel. Add the color marker dye to the DNA ladder.

_ 4 5

_ 6

DNA bands under UV light

Factors Affecting Migration

The most impt factor is the length of the DNA molecule, smaller molecules travel further. Increase the agarose conc of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution .

Visualisation : EtBr and Dyes

The most common dye used for AGE is ethidium bromide. It fluoresces under uv light when intercalated into DNA. By running DNA through EtBr-treated gel and visualizing it with UV light , distant bands of DNA become visible . Alternative dye such as SYBR Green may be used.

Loading buffers area added with DNA in order to visualize it and sediment it in the gel. Xylene cynaol and Bromophenol blue are used. Other less frequently used markers are Cresol red and Orange G.

Uses

In restriction mapping of cloned DNA Analysis of PCR products ,eg. In molecular genetic diagnosis or genetic fingerprinting Separation of restricted genomic DNA prior to southern transfer, or RNA prior to northern transfer.

The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered. The disadvantages are the gel can melt during electrophoresis, the buffer can become exhausted and different forms of genetic material may run in unpredictable forms.

Chromatography

It was the Russian botanist Mikhail Semyonovich Tscet who invented the first chromatography technique in 1900 during research on chlorophyll.

Column chromatograhy is one of the most common methods of protein purification. In this method a protein is passed through a column that is designed to trap or slow up the passing of proteins based on a particular property such as size, charge or composition.

Some of the more common column are 1. Ion exchange chromatography 2. Hydrophobic interaction column 3. Affinity chromatography. 4. Gel filtration ( size exclusion ) chromatography.

Ion Exchange Chromatography

This method is based on the charge of the protein. If the protein to be studied has a positive charge, then it has to pass through a column of negative charge. The negative charge on the column will bind the positively charged protein, and other protein will pass through the column. Then salting out is done to release the positively charged protein from the negatively charged column.

The column that does this is called cation exchange column and often uses sulfonated residues. Likewise negatively charged protein can bind to a positively charge column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting Out

This is a procedure which will release the protein from the column. This technique uses a high salt concentration solution. The column has a higher attraction for the charge of salts than for the charged protein, and it will release the protein in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt. Different proteins elute at different salt conc.

Basic steps in ionic Exchange

Prep the Column. Pour the buffer over the column to make sure is has equilibrated to the required pH. Load protein solution. Salt out. Increase the salt conc to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. Remove salts. Use dialysis to remove the salts from the protein solution.

Hydrophobic interaction chromatography

HIC uses the hydrophobic properties of some proteins. Hydrophobic groups on the protein bind to hydrophilic groups on the column. The more hydrophobic a protein is, the stronger it will bind to the column.

Load the proteins in the presence of a high conc of ammonium sulfate. Ammonium sulfate is a chaotrophic agent. It increases the chaos in water, and thereby increases hydrophobic interactions. Ammonium sulfate also stabilizes proteins. So the result of using HIC column the protein is in its stable form.

The hydrophobic column is packed with a phenyl agarose matrix. In the presence of high salt conc the phenyl groups on this matrix binds hydrophobic portions of proteins.

Affinity Chromatography

This relies in the biological functions of a protein to bind it to a column. The most common type involves a ligand , a specific small biomolecule. This small molecule is immobilized and attached to a column matrix, such as cellulose or polyacrylamide. The target protein is then passed through the column and bound to by its ligand, while other proteins elute out.

Elution of the target protein is usually done by passing through the column a solution that has in it a high conc of free ligand. This is a very efficient purification method since it relies on the biological specificity of the target protein , such as the affinity of an enzyme for a substrate.

Gel Filtration Chromatography

Gel filtration, or size exclusion, chromatography separates proteins on the basis of their size. The column packed with a matrix of fine porous beads.

It works somewhat like a sieve, but in reverse. The beads have in them very small holes. As the protein solution is poured on the column, small molecules enter the pores in the beads. Larger molecules are excluded from the holes, and pass quickly between the beads.

The larger molecules are eluted first. The smaller molecules have a longer path to travel, as they get stuck over and over again in the maze of pores running from bead to bead. These smaller molecules, therefore, take longer to make their way through the column and eluted last

Load proteins into column

Smaller molecules enter the channels in the beads and have to travel farther

Larger molecules travel between beads and elute first

Probes

Probes are complimentary sequences of nucleotide bases to the specific mRNA sequence of interest. These probes can be as small as 20 40 base pairs or be up to 1000bp.

Types of Probes

1. 2. 3. 4.

There are essentially four types of probes used. Oligonucleotide probe Single stranded DNA probe Double stranded DNA probe RNA probe

1.

Oligonucleotide probes. : these are produced synthetically by a automated chemical synthesis. This method utilizes deoxynucleotides, which requires designing the sequence of the probe when using oligonucleotide probes. These probes have the advantage of being resistant to RNases and are small, generally around 40 50 base pairs.

This is ideal for in situ hybridization because their small size allows for easy penetration into the cells or tissue of interest. In addition, because they are synthetically designed, it is possible to make a series of probes that have the same GC content.

Single Stranded DNA Probes

These are much larger 200 -500 bp size range. They can be produced by reverse transcription of RNA or by amplified primer extension of a PCRgenerated fragment in the presence of a single antisense primer.

That is once amplified sequence of interest is identified a subsequent round of PCR is carried out using the first PCR product template, but only using anti-sense primers, thus producing single stranded DNA. They require time to prepare, expensive reagents are used, and a good repertoire of molecular skills are required.

Double Stranded DNA Probe

These can be produced by the inclusion of the sequence of interest, which is replicated, lysed. And the DNA extracted, purified and the sequence of interest is excised with restriction enzymes.

The advantage is that it is possible to obtain large quantities of the probe sequence. These probes are generally less sensitive because of the DNA strands to rehybridize to each other are not widely used.

RNA Probes

They have an advantage that RNARNA hybrides are very thermostable and are resistant to digestion by RNases. There are 2 methods of preparing RNA probes

1.

2.

Complimentary RNAs are prepared by an RNA polymerase-catalysed transcription of mRNA. Alternatively, in vitro transcription of linearized plasmid DNA with polymerase can be used to produce the RNA probes. Here plasmid vectors containing polymerase from bacteriophages T3, T7 or SP6 are used.

These probes are very sensitive to RNases and so scrupulous sterile technique is observed, or these probes can be destroyed easily. They still widely used with in situ hybridization.