Question

177 4. Prepare a brief written procedure for each of the biological/genetic test(s) that will be performed. Describe observations that indicate a positive test result Note: DNA isolation, PCR, and RE digestion will be performed by the Laboratory Technician. Include only sample preparation steps for these methods.

Answer 1

DNA isolation

1. Preparation of a cell extract:

To extract DNA from a tissue/cells of interest, in this experiment the heart muscle of rats, the cells have to be separated and the cell membranes have to be disrupted. The "Extraction buffer" helps in carrying out these processes. Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes Mg2+ ions that are essential for preserving the overall structure of the cell membrane, and SDS (Sodium Dodecyl Sulfate) which aids in disrupting the cell membranes by removing the lipids of the cell membranes are included in the extraction buffer. Having lysed the cells, the final step in the preparation of a cell extract is removal of insoluble cell debris. Cell debris and partially digested organelles etc. can be pelleted by centrifugation leaving the cell extract as a reasonably clear supernatant.

2. Purification of DNA from cell extract

In addition to DNA the cell extract will contain significant quantities of protein and RNA. A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form. The standard way to de-proteinize a cell extract is to add phenol or a 1:1 mixture of phenol:chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solutions. The aqueous solution of nucleic acid can be removed with a pipette. The effective way to remove RNA is with the enzyme ribonuclease, which will rapidly degrade these molecules into ribonucleotide subunits.

3. Concentration of DNA samples

The most frequently used method of concentration is ethanol precipitation. In the presence of salt and at a temperature of -20 °C or less, absolute ethanol will efficiently precipitate polymeric nucleic acids. With a concentrated solution of DNA one can use a glass rod to pull out the adhering DNA strands while for dilute solutions precipitated DNA can be collected by centrifugation and redissolving in an appropriate volume of water.

4. Measurement of purity and DNA concentration

DNA concentrations can be accurately measured by UV absorbance spectrometry. The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA sample. Usually absorbance is measured at 260 nm, at which wave length an absorbance of 1.0 corresponds to 50 µg of double-stranded DNA per ml. UV absorbance can also be used to check the purity of a DNA preparation. With a pure sample of DNA the ratio of the absorbancies at 260 nm and 280 nm (A260/A280) is 1.8. Ratios of less than 1.8 indicate that the preparation is contaminated, either with protein or with phenol.

Observation

Today there are many specialised extraction methods. These are generally either solution-based or column-based. The extraction of DNA has become much easier with the emergence of commercial kits and the automation of the process. Such changes have both sped up production and increased the yield of DNA.

PCR

The optimal conditions for the concentration of Taq DNA polymerase, template DNA, primers, and MgCl2 will depend on the system being utilized. It may be necessary to determine the optimal conditions for each individual component. This is especially true for the Taq DNA polymerase, cycling parameters, and the MgCl2 concentration. It is recommended the enzyme and the MgCl2 be titrated to determine the optimal efficiency.

1. Add the reagents to an appropriately sized tube in the order provided in the table. (Select appropriate table for reaction setup: standard or readymix reagent.) For a large number of reactions, a mastermix without the template should be set up and aliquoted into reaction tubes. At the end, template should be added to appropriate tubes.

2.Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.

Note: Add 50 µL of mineral oil to the top of each tube to prevent evaporation if using a thermal cycler without a heated lid.

3.Amplify. The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.

4.The amplified DNA can be evaluated by agarose gel electrophoresis and subsequent ethidium bromide staining.

Note: Mineral oil overlay may be removed by a single chloroform extraction (1:1), recovering the aqueous phase.

Observation

To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis may be employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder, a molecular weight marker which contains DNA fragments of known size run on the gel alongside the PCR products.

RE digestion

1.Add components to a clean tube in the order shown:

1 µL DNA (concentration 1 µg/µL)

2 µL 10x buffer

1 µL restriction enzyme

16 µL sterile water

2.Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.

3.Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.

4.The digested DNA is ready for use in research applications.

Observation

Bacteriophage λ is a virus that attacks bacterial cells and is one of the most studied viruses. The information from the relatively simple virus genomes has been used to test theories and develop concepts that apply to the genetics of living organisms. The DNA of Bacteriophage λ is approximately 48,514 base pairs or 48.514 kilobase pairs in length while the human genome is approximately 3 billion base pairs.

This experiment uses special “restriction” enzymes that act as chemical scissors to cut λ DNA into pieces. Each enzyme recognizes a unique sequence of 4-6 bases along the DNA strand and cuts the strand at these sites - the first step in a process called restriction mapping. These smaller, specific sections of an organism’s DNA can then be studied in detail and an outline of the whole genome can be constructed. This procedure is one of the most important in modern biology.

The small fragments of DNA are separated by gel electrophoresis. The movement of the fragments will always be towards the positive electrode because DNA is a negatively charged molecule. The fragments move through the gel at a rate that is determined by their size and shape, with the smallest moving the fastest.

DNA cannot be seen as it moves through the gel. A loading dye must be added to each of the samples before it is pipetted into the wells. The progress of the dye can be seen in the gel. It will initially appear as a blue band, eventually resolving into two bands of different colors.

The faster moving, purplish band is bromophenol blue dye that migrates at roughly the same rate as a 300 base pair fragment of DNA. The slower moving aqua band is xylene cyanol, nearly equivalent to a 9000 base pair fragment. The faster moving band must move at least 4-7 cm from the wells to achieve the best separation of DNA for analysis. Care should be taken not to let the bromophenol blue band run off the end of the gel.