eChapters

PCR Principles and Procedure

Polymerase chain reaction (PCR) is a technique that allows the generation of large amounts of a single DNA sequence from a mixture of sequences; the fragment generated can be designed to contain specific starting and ending positions based on the needs of the experiment. PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. 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:

Cell Genotypes

A large variety of bacterial strains, like E. coli, are frequently used in molecular biological experiments. Students should choose the strain or strains required for an experiment on the basis of genetic properties of the strains. It is thus pertinent that a sound knowledge of the genetic makeup of the E. coli stains is acquired.

The genetic makeup of the strains of E.coli is distinctly different from the wild type (K12 or B strains) strains from which they are derived and contains genes that can be modified. Genes are supposed to be mutated when they are not listed in a genotype. In most cases, the genes listed are mutated to the point of inactivity. In a few cases, a “-” (indicating inactive or absent or deleted), “+” (indicating active). , The genotype is a factor in choosing a bacterial strain. Chosen strains should be ascertained for their utility for the specific experiment.

Small Scale Cultures

Fresh culture from a freshly streaked plate should be taken while experimenting with E. coli cells. For preparing a small scale culture of E. coli , prepare 3-5 ml of LB medium/broth (or appropriate broth include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. Inoculate one tube with a single colony from a fresh plate. The second tube is used as a broth control. Incubate both tubes at 37°C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Never allow cells to overgrow. For some applications, cells can be stored at 4°C for short periods prior to use.

Storage

Nucleic acids are very sensitive to contaminations hence care should be taken while processing and storing DNA and RNA. The following properties of reagents and conditions need to be taken care of:

Heavy metals cause breakage of phosphodiester linkage. One can use EDTA which is an excellent heavy metal chelator.

UV rays cause extensive damage to nucleic acids. UV light at 260 nm causes breakage of thymine dimers and cross-link, including loss of biological activities.

Ethidium bromide results in photo oxidation of DNA. Oxidation products can cause phosphodiester breakage.

Nucleases are found on human skin; therefore, it is advisable to avoid direct or indirect contact between nucleic acids and fingers.

Most DNAes are not very stable; however, many RNAses are very stable and can absorb to glass or plastic and remain active.

At -20°C: extensive single and double strand breaks results. Hence for long-term storage it should be stores at -70°C. ? It is best to store DNA in high salt ( >1M) in the presence of high EDTA (>10mM) at pH 8.5, for long-term storage.

Storage of DNA in CsCl with ethidium bromide in the dark is an excellent storage medium. There is about one phosphodiester break per 200 kb of DNA per year.

Storage of ë DNA in the phage is better than storing the pure DNA.

Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids and solids should be autoclaved separately. Ensure that the lids on all bottles are loose. Do not put a large number of items in tray, to ensure that all items reach the appropriate temperature. Also allow sufficient air/steam circulation.

Steps in autoclave

Pressure should read ‘O’ when you open the pressure chamber of the autoclave.

Place items to be autoclaved in the autoclave and close the door. Some autoclaves require that you also lock the door after it’s closed.

Set time - typically 20 minutes.

Temperature should be set at 121°C already, but double-check and change if necessary.

Set cycle: If liquid, set “liquid cycle” or “slow exhaust”. If dry, set “dry cycle” or “fast exhaust” + dry time.

Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to “sterilize”

Several substrates, like proteins (enzymes), are very sensitive to alteration in the concentration of the solution they are placed in. Hence is the importance of a buffer which controls the chemical properties of the medium. Literally a “buffer” has the ability to resist changes in the hydrogen ion concentration. Although, buffers do contain other molecules, that might, in addition to the hydrogen ion concentration, influence the ionic strength, the activity of enzymes, and other parameters of the experiment.

In any biological experiment, appropriate buffer must be selected based on their effect on the experiment. Ideal buffers control pH and ionic strength without interacting adversely with the experiment undertaken. For example, although phosphate is a common buffer, it may not be suited when phosphate is a substrate or product of the reaction being studied. Also, some proteins interact poorly with some buffers. For example, Tris is not ideal as it exhibit high pKa and the large change in pKa when temperature changes, although it is cheap and most proteins are stable in Tris buffers, moreover it seldom reacts with reaction substrates hence, Tris buffer is commonly used in biochemical experiments.

To prepare a series of dilutions of the original more concentrated stock solution, one need to consider both the required final concentration and required volume of the diluted material. The dilution can be readily calculated by the equation:

A spectrophotometer is an analytical instrument that measures the absorbance of a test sample (solution). The term “spectroscopy” comes from the word “spectrum”. “Spectroscopy” therefore implies the use of multiple wavelengths of light. Spectrophotometers have the ability to specifically measure absorbance at specific wavelengths. The most commonly used method to allow this involves a “monochromator”, a device (either a prism, or more commonly, a diffraction grating) that splits the incident light into its component wavelengths, and allows only light of the desired wavelength to reach the sample. The ability to measure absorbance at different wavelengths is very useful, because the extinction coefficient of a compound varies with wavelength. In addition, the absorbance spectrum of a compound can vary depending on the chemical composition of the compound, and depending on the nature of the solvent around the compound.

Absorbance is a useful quantity and follows the Beer-Lambert law. The extinction coefficient of a molecule at a given wavelength can be calculated using the Beer-Lambert equation from absorbance measurements for solutions of known concentration, which states that:

In molecular biology and biochemistry, the ability to accurately and reproducibly measure and transfer small volumes of liquids is critical for obtaining useful results. For volumes less than 1 ml, the most common method for measuring liquid volumes involves the use of a device known as a pipetman or micro-pipette.

The pipetmen can be both fixed and variable volumes. Commonly used pipetmen are: P1000, P200, P20, P10 etc. P1000 are useful for volumes from 200 to 1000 µl. P200 are useful for volumes from 20 to 200 µl. P20 are useful for volumes from 0.5 to 20 µl. For beginners fixed volume pipetmen are more useful. It is important to ascertain whether the correct pipetman is chosen for measuring the volume one needs.

This book is designed to introduce to the students some of the most widely used experimental procedures in biotechnology, including DNA/RNA isolation, manipulation, cloning, protein purification and characterization. The students will also get familiarized with some of the equipments frequently used in biochemistry and molecular biology.

The biotechnology laboratory course is an exploration of procedures. Hence, in order to get full benefit from the course, the students need to read the manual, which is self explanatory. The more effort they put into the course work, the more they will learn. Before the start of actual lab works, one needs to spend some time reading the Laboratory Manual. This reading will provide background information and an outline of the procedures to be performed.

The biotechnology laboratory is conducted as a “directed” research project. This means that although the general procedures are well established, the overall goal of each experiment is the acquisition of new information. Because of the nature of scientific research, predicting the outcome of experiments that have not previously been performed is difficult. It may therefore be necessary to design new experiments based on the results of previous ones, or to repeat experiments that yielded uninterruptable or ambiguous results.

Plant hormones - They are defined as:

1. Small
2. Organic compounds;
3. Synthesized by the plant;
4. Active in low concentration (<10^-6);
5. Promote or inhibit growth and developmental responses;
6. Often show a separation of the site of production and the site of action (although this isn’t as clear a distinction as in animals).

Based on these criteria, would the following substances be considered hormones? Ca²⁺, sucrose, 2 4 - D, glycine, or K⁺?

PLANT NUTRITION

Plants use inorganic minerals for nutrition, whether grown in the field or in a container. Complex interactions involving weathering of rock minerals, decaying organic matter, animals, and microbes take place to form inorganic minerals in soil. Roots absorb mineral nutrients as ions in soil water. Many factors influence nutrient uptake for plants. Ions can be readily available to roots or could be “tied up” by other elements or the soil itself. Soil too high in pH (alkaline) or too low (acid) makes minerals unavailable to plants.

I. Definition

Solute transport in plants, translocation, primarily occurs in the phloem, but it can occur in the xylem.

II. Solute Transport in the Xylem

Some solutes are transported in the xylem

Water and dissolved ions are the main substances in vessels/tracheids

These materials are transported via transpiration stream

Xylem sap may also contain organic materials, usually in relatively low concentration (with a notable exception being maple sap in the spring which is comprised of 2% or more sucrose).

Substances move at different rates depending on matrix effects, metabolic needs, etc.

I. Definitions

Transpiration - evaporation of water from a plant surface

Evapotranspiration - evaporation of water from a plant surface and the soil (and abiotic surroundings.

Take-home-lesson: Plants loose a lot of water by transpiration. See text for some specific examples.

Water, of its very nature, as it occurs automatically in the process of cosmic evolution, is fit, with a fitness no less marvelous and varied than that fitness of the organism which has been won by the process of adaptation in the course of organic evolution.

WATER IS ABSOLUTELY ESSENTIAL FOR ALL LIVING ORGANISMS

The evidence:

Most organisms are comprised of at least 70% or more water. Some plants, like a head of lettuce, are made up of nearly 95% water;

When organisms go dormant, they loose most of their water. For example, seeds and buds are typically less than 10% water, as are desiccated rotifers, nematodes and yeast cells;

Earth is the water planet (that’s why astronomers get so excited about finding water in space).

Water is the limiting resource for crop productivity in most agricultural systems.

WHAT IS PLANT?

A. Definition - by most definitions, a plant:

is multi-cellular;

is non-motile

has eukaryotic cells

has cell walls comprised of cellulose

is autotrophic; and

exhibits alternation of generations - has a distinctive diploid (sporophyte) and haploid (gametophyte) phase.

The polymerase chain reaction (PCR) is a rapid way of amplifying (duplicating) specific DNA sequences

Method was devised by Kary Mullis of Cetus Corporation, Emeryville He recieved a $20,000 bonus and later a Nobel Prize

DNA heated to high temperature is not destroyed; separates into single strands, but reforms helix when cooled

PCR Method:

DNA to be amplified is put into solution containing:

Restriction enzymes cut only at specific sequences of 4 to 8 bases

A long restriction site is rare. Cutting rare sites will produce a few large pieces of DNA.

Restriction enzymes with long sites → small number of large fragments

Restriction enzymes with short sites → large number of small fragments

BACTERIA HAVE SMALL, COMPACT GENOMES

We now know the complete DNA sequences for about a dozen bacteria

Bacteria seem to need about 500 to 5000 genes to conduct their lives

Because they are small, bacterial genomes are highly organized

Most of the time there are only a few bases between the end of one gene and the beginning of the next

Few introns are found in bacteria

Bacteria contain accessory “chromosomes” called plasmids; each has only a few genes

Viruses are much simpler than cells

Protein capsid

Genetic information stored on DNA or RNA:

Types of genome:

Double strand DNA: sometimes circular

Single strand DNA: sometimes circular

Double strand RNA

Single strand RNA

Some can be crystallized

DNA replication. The double helix is unwound and each strand acts as a template. Bases are matched to synthesize the new partner strands.

DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is “replication” in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Therefore, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule. Cellular proofreading and error toe-checking mechanisms ensure near perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations in the genome, called “origins”.

Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.