eChapters

Introduction

The AFLP amplified fragment polymorphism also known as selective restriction fragment amplification (SRFA) technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously. The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an isolate or the degree of similarity among isolates. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry. produces highly complex DNA profiles by arbitrary amplification of restriction fragments ligated to double-stranded adaptors with hemi-specific primers harboring adaptor-complementary 5' termini.The technique has been widely used in the construction of genetic maps containing high densities of DNA marker loci.

The AFLP protocol amplifies restriction fragments obtained by endonuclease digestion of target DNA using “universal” AFLP primers complementary to the restriction site and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain selective nucleotides at the 3' termini that extend into the amplified restriction fragments. These arbitrary terminal sequences result in the amplification of only a small subset of possible restriction fragments. The number of amplified fragments can therefore be “tailored” by extending the number of arbitrary nucleotides added to the primer termini. Alternatively, the use of endonuclease combinations that vary in their restriction frequency can also be used to tune the number of amplicons. Generally, the abundant restriction fragments produced from complex genomes require of AFLP primers with longer selective regions. Conversely, analysis of small genomes require of only few arbitrary nucleotides added at the primer 3' termini. The resulting AFLP fingerprints are usually a rich source of DNA polymorphisms that can be used in mapping and general fingerprinting endeavors.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

Introduction

This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors.

Principle

The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

DNA can be easily isolated and purified after size selection on an agarose gel. The fragment of interest is simply cut out of the gel with a razor blade and purified by a number of different methods.The easiest is to use a method that involves first dissolving the agarose slice in a solution at 50°C, then binding the DNA from the melted agarose to a silica-gel membrane.

Principle

In this method following gel electrophoresis using low gelling/ melting temperature agarose, the band of interest is removed from the gel. The agarose slice is then melted and subjected to phenol extraction.

Introduction

The primary tools used by the molecular biologists in manipulating DNA are restriction enzymes and other DNA/RNA modifying enzymes. Over the years, the number and uses of these enzymes have increased as new molecules have been discovered. Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA. They are present in bacteria presumably to destroy DNA from foreign sources. By cleaving the foreign DNA at specific sites. The host bacterial DNA os protected from cleavage because specific recognition sites are modified, usually by methylation form methylase at one of the bases in the site, making the site no longer a substrate for RE cleavage. Host bacteria used to propagate cloned DNA in the laboratory arte usually mutant in the host restriction genes, thus their intracellular enzyme activities will not destroy the foreign recombinant sequences. The endonuclaase with its accompanying methylase is called a restriction modification (R-M) system.

At least four different kinds of R-M systems exist characterized on the basis of the subunit composition, cofactor requirements and type of DNA cleavage. Most characterized enzymes belong to type II class, together with type IIS class. They comprise the commercially available restriction enzymes used for DNA analysis and manipulation. Type I and Type III enzymes are relatively uncommon and a few additional enzymes fit none of the classes.

Among more than 3000 different enzymes isolated from bacterial strains many share common specificities. Restriction enzymes that recognize identical sequences have been called as isoschizomers.

History of PCR

A 1971 Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F) environments) paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

Developed the PCR in 1984 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

Introduction

Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. SDS-PAGE is the most widely used method for qualitatively analyzing any protein mixtures. The method is based on the separation of proteins according to their size and then locating by binding them to a dye. It is particularly useful for monitoring protein purity and determines their relative mass. The SDS-PAGE enables the students to understand the theory behind the techniques in checking the purity of the protein and identifying the protein in a test sample.

Separation of Proteins

Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e.: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

Electrophoresis is a method of separating out large molecule ( DNA or protein). An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins.

What is Gel Electrophoresis?

A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Principle

In this method the estimation of pentose sugar is carried out. Acid hydrolysis of RNA releases ribose (a pentose sugar) which in the presence of strong acid undergoes dehydration to yield furfural. Orcinol, in the presence of ferric chloride as a catalyst, reacts with furfural producing a green colored compound which can be quantitatively estimated at A_{665 nm}. DNA gives a limited positive reaction with Orcinol test.

Materials and Reagents

1. Spectrophotometer
2. Boiling water bath
3. 5% chloroform (HClO₄)
4. Standard RNA solution : Dissolve yeast RNA (500ìg/ml) in 5% HClO₄ . make different dilutions to obtain solutions containing 100 – 500ìg RNA/ml with 5% HClO₄ .
5. Orcinol reagent : Dissolve 100mg of ferric chloride (FeCl₃.6H₂O) in 100 ml of concentration HCl and then add 3.5 ml of 6% solution of Orcinol prepared in alcohol.

To Estimate the DNA Using Diphenylamine

Introduction

DNA estimation is helpful to know about the total amount of DNA of prokaryotic as well as eukaryotic system. One method used for DNA estimation is Diphenylamine assay, is that of Burton who modified the original procedure of Dische. This procedure is based on treatment of nucleic acid with hot perchloric acid to hydrolyze DNA nucleotides. Diphenylamine reacts specifically with deoxyribose in DNA to form a blue-colored complex. Since diphenylamine does not react with ribose, one can assay for DNA in the presence of contaminating RNA.

Principle

The DPA method is based on the colorimetry measurement of products formed by the reaction of ù- hydroxylevulinaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 2.5 N perchloric acid.

When DNA is treated with diphenylamine under acidic condition (PCA) a blue complex is formed with a sharp absorption maximum at 595 nm (600 nm). In acid solution, the straight chain form of the deoxypentose is converted to highly reactive ω – hydroxylevulinaldehyde that reacts with diphenylamine to give blue complex. The reaction yields two mixture of product which together have absorbance maximum at 595 nm (600 nm). Interferences include proteins, lipids, saccharides and RNA. In DNA the only deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present.

Introduction

DNA denaturation, also called DNA melting, is the process by which double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands during the breaking of hydrogen bonding between the bases. Both terms are used to refer to the process as it occurs when a mixture is heated, although “denaturation” can also refer to the separation of DNA strands induced by chemicals like urea. For multiple copies of DNA molecules, the melting temperature (T_m) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the “random-coil” states. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.

Applications of DNA Denaturation

The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenosine / thymine base-pairing, the amount of cytosine and guanine in a genome (called the “GC content”) can be estimated by measuring the temperature at which the genomic DNA melts. Elevated temperatures are associated with high GC content. GC ratios within a genome are found to be markedly variable. These variations in GC ratio within a genome of higher organisms results in a mosaic like formation with islet regions called isochores. This results in the variations in staining intensity in the chromosomes. The isochores include in them essential protein coding genes, termed housekeeping genes and thus determination of ratio of these specific regions contributes in mapping these essential genesbacterial systematics has recommended use of GC ratios in higher level hierarchical classification. For example, the Actinobacteria are characterised as “high GC-content bacteria”. In Streptomyces coelicolor A3(2), GC content is 72%. The GC-content of Yeast (Saccharomyces cerevisiae) is 38%, and that of another common model organism Thale Cress is 36%. Because of the nature of the genetic code, it is virtually impossible for an organism to have a genome with a GC-content approaching either 0% or 100%. A species with an extremely low GC-content is Plasmodium falciparum (GC% = ~20%), and it is usually common to refer to such examples as being AT-rich instead of GC-poor.

Procedure

Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube.

Heat the tube in a boiling water bath for 1 hour.

Cool the tube, open it and place the contents into a centrifuge tube. Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA.

Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography.

Using a micropipette, spot 20 µl of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than one large drop). Allow the spots to completely dry before proceeding.

Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20 cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40° C for about 2 hours.

Locate the spots of nucleotides by their fluorescence under an ultra-violet light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid and finally, uridylic acid.

After cafefully marking the spots, cut them out with cutters and place the paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add 5.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

DNA Migration in Agarose and Polyacrylamide Gels

Recommended Gel Percentages for Separation of Linear DNA

Mole (mol)

1 mole of any substance contains 6.02 x 10²³ units. The number of moles in a given weight of substance can be calculated using following equation.

1. pH Meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

2. UV Spectrophotometer

Perhaps there is no other technique except UV-visible spectroscopy, which has been so extensively employed and has such a profound impact on progress in biochemical investigations. UV-visible spectroscopy has undoubtedly become an indispensable tool and is routinely used for identification and quantitative estimation of most of the biomolecules. Besides other applications, most of the enzyme assays and the screening of biomolecules recovered in different fractions by separation processes such as column chromatography, density gradient centrifugation etc, rely heavily on spectrophotometer or colorimetric technique.

Search for genetic material—nucleic acid or protein/DNA or RNA?

• Griffith’s Transformation Experiment
• Avery’s Transformation Experiment
• Hershey-Chase Bacteriophage Experiment
• Tobacco Mosaic Virus (TMV) Experiment

Timeline of Events

• 1890 Weismann - substance in the cell nuclei controls development.
• 1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.
• 1928 Griffith’s Transformation Experiment
• 1944 Avery’s Transformation Experiment
• 1953 Hershey-Chase Bacteriophage Experiment
• 1953 Watson & Crick propose double-helix model of DNA
• 1956 Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material

BC

1750 The Sumerians brew beer.

500 The Chinese use moldy soybean curds as an antibiotic to treat boils.

250 The Greeks practice crop rotation to maximize soil fertility.

100 Powdered chrysanthemum is used in China as an insecticide.

AD: Before the 20^th Century

1590 The microscope is invented by Janssen.

1663 Cells are first described by Robert Hooke.

1675 Leeuwenhoek discovers protozoa and bacteria.

1745 Maupertuis proposes an adaptationist account of organic design

1797 Jenner inoculates a child with a viral vaccine to protect him from smallpox.

1802 The word “biology” first appears.