DNA Analysis

DNA Analysis
By Kristy Annely

The DNA of a person is responsible for providing information regarding who and what the person is since it contains the design of the genetically passed traits taken from both parents. A lot of information may be attained when one carefully studies the DNA and relates it to possible other discoveries about the person, such as parentage, disease predisposition, etc. DNA is responsible for the person's hair color, eye color, build, susceptibility to certain disorders, and a lot more. Careful analysis must be done in order to use the DNA as a means to study and evaluate a person with regard to inherent traits passed on by his/her parents.

DNA analysis entails the careful scrutiny of the DNA. Understanding how the DNA works, its physical structure, the limits of its characteristics, and how these all help in providing much needed personal information are some of the ways in which DNA properties are optimized. Not everyone can successfully and properly analyze DNA. Aside from years of study devoted to the understanding of DNA, DNA analysts also go through years of experience and practice in laboratories and in the practice of DNA testing and analysis to enable them to analyze DNA and how it works. Training of DNA analysts also includes understanding sophisticated tools and machines designed for specific DNA analysis. Proper management and use of these devices would make them more efficient in the effort to analyze DNA correctly.

Most DNA consists of 23 pairs of chromosomes, the mitochondrial DNA inherited from the mother, and red blood cells. All these elements are carefully purified, studied, and then measured against existing DNA criteria, depending on which use the DNA analysis is deemed for. DNA analysis determines the sequence of the base pairs along the length of the DNA and will provide information as to which of the gene components contains just genes, which regulate genes, which among them have functions, or which have functions still to be discovered.

Several commonly used DNA analysis techniques include: digestion by restriction endonucleases, gel electrophoresis, blotting and hybridization, synthesis of nucleic acids through polymerase, synthesis of nucleic acid probes, nucleotide sequencing, molecular cloning, or the analysis of reporter genes.

DNA provides detailed information on DNA, DNA Testing, DNA Structures, DNA Fingerprinting and more. DNA is affiliated with Free DNA Testing.

Article Source: http://EzineArticles.com/?expert=Kristy_Annely
http://EzineArticles.com/?DNA-Analysis&id=353545

dna analysis information

Procedure Isolation and Purification of Total Genomic DNA from a bacteria.

This will be a small scale isolation and purification, providing DNA of sufficient purity for use as template in a PCR reaction.

wear goggles and gloves

discard reagents and tubes in labeled waste containers

1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.

2. Centrifuge at 15,000 × g for 2 minutes to pellet the cells.

Place your tube opposite that of another student to balance to rotor.

Do not initiate a spin cycle until the rotor is fully loaded; this minimizes the total number of runs required.

3. Transfer the supernatant back into the culture tube it came from and discard this culture tube as biohazard waste.

Carefully remove as much of the supernatant as you can without disturbing the cell pellet. The pellet may be on the side of the tube, not squarely on the bottom.

4. Resuspend the cell pellet in 600µl of Lysis Solution (LS) and then incubate at 80°C for 5 minutes to lyse the cells.

Gently pipet until the cells are thoroughly resuspended.

LS contains the anionic detergent sodium dodecyl sulphate (SDS).

5. Cool the tube contents to room temperature.

Do not rely on temperature equilibration with ambient air. Place the tube in a room temperature water bath for several minutes.

6. Add 3µl of RNase solution to the cell lysate. Invert the tube 2–5 times to mix.

7. Incubate at 37°C for 15–60 minutes to digest RNA. Cool the sample to room temperature.

8. Add 200 µl of Protein Precipitation Solution (PPS) to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds to mix.

9. Incubate the sample in an ice/water slurry for 5 minutes.

10. Centrifuge at 15,000 × g for 3 minutes.

11. Transfer the supernatant (≤800 µl) containing the DNA to a clean 1.5ml microcentrifuge tube containing 600µl of room temperature isopropanol (IPA).

Be sure that you don’t suck up and transfer any of the grungy precipitate.

12. Gently mix by inversion. The DNA should come out of solution as visible thread-like strands.

13. Centrifuge at 15,000 × g for 2 minutes.

14. Carefully pour off the supernatant (do not pipette) and drain the tube on clean absorbent paper.

The DNA pellet may not be visible.

Do not allow the DNA pellet to dry out.

15. Add 600µl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.

16. Centrifuge at 15,000 × g for 2 minutes.

17. Carefully pour off the ethanol (do not pipette) and drain the tube on clean absorbent paper.

18. Allow the pellet to air-dry for 10–15 minutes.

You want to evaporate as much of the ethanol as possible without letting the DNA pellet completely dry.

19. Add 100µl of DNA Rehydration Solution (RH) to the tube and rehydrate the DNA by incubating at 65°C for 1 hour.

Periodically mix the solution by gently tapping the tube.

Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or at 4°C, preferably on a low speed shaker.

20. Store the DNA at 2–8°C

DNA ISOLATION AND PURIFICATION METHODS

Cesium chloride density gradients

Genomic DNA can be purified by centrifugation through a cesium chloride (CsCl) density gradient. Cells are lysed using a detergent, and the lysate is alcohol precipitated. Resuspended DNA is mixed with CsCl and ethidium bromide and centrifuged for several hours. The DNA band is collected from the centrifuge tube, extracted with isopropanol to remove the ethidium bromide, and then precipitated with ethanol to recover the DNA. This method allows the isolation of high-quality DNA, but is time consuming, labor intensive, and expensive (an ultracentrifuge is required), making it inappropriate for routine use. This method uses toxic chemicals and is also impossible to automate.

Anion-exchange methods

Solid-phase anion-exchange chromatography is based on the interaction between the negatively charged phosphates of the nucleic acid and positively charged surface molecules on the substrate. DNA binds to the substrate under low-salt conditions, impurities such as RNA, cellular proteins, and metabolites are washed away using medium-salt buffers, and high-quality DNA is eluted using a high-salt buffer. The eluted DNA is recovered by alcohol precipitation, and is suitable for all downstream applications. Anion-exchange technology completely avoids the use of toxic substances, and can be used for different throughput requirements as well as for different scales of purification. The isolated DNA is sized up to 150 kb, with an average length of 50–100 kb.

Silica-based methods — DNeasy Tissue Kits

DNeasy Tissue technology provides a simple, reliable, fast, and inexpensive method for isolation of high-quality DNA. This method is based on the selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts. Use of optimized buffers in the lysis procedure ensures that only DNA is adsorbed while cellular proteins, and metabolites remain in solution and are subsequently washed away. This is simpler and more effective than other methods where precipitation or extraction is required. Ready-to-use DNA is then eluted from the silica-gel membrane using a low-salt buffer. No alcohol precipitation is required, and resuspension of the DNA, which is often difficult if the DNA has been over-dried, is not required. DNeasy Tissue Kits are designed for rapid isolation of pure total DNA (genomic, viral, and mitochondrial) from a wide variety of sample sources, including fresh and frozen animal cells and tissues, yeasts, and blood. DNA purified using DNeasy Tissue Kits is free from contamination and enzyme inhibitors and is highly suited for applications such as Southern blotting, PCR, real-time PCR, RAPD, RFLP, and AFLP analyses. DNeasy Tissue Kits are available in convenient spin-column or 96-well formats, suitable for a wide range of throughput needs. Genomic DNA isolated using DNeasy Tissue technology is up to 50 kb in size, with an average length of 20–30 kb. DNA of this length is particularly suitable for PCR analysis as well as Southern blotting analysis. Silica-gel spin technology is not suitable if genomic DNA >50 kb is required for certain cloning or blotting applications. The DNeasy Tissue procedure is suitable for both very small and large sample sizes, from as little as 100 cells up to 5 x 106 cells. In order to obtain optimal DNA yield and quality, it is important not to overload the DNeasy System, as this can lead to significantly lower yields than expected Overloading the DNeasy System can also adversely affect the purity of the DNA The DNeasy Tissue procedure is also highly suited for purification of DNA from very small amounts of starting material. If the sample has less than 5 ng DNA (<10,000 style=""> (a homopolymer such as poly dA, poly dT, or gDNA) should be added to the starting material. Ensure that the carrier DNA does not interfere with the downstream application.

Reference:

www1.qiagen.com/literature/brochures/Gen_DNA_Pur/1019469_BROS_DNYTi_p10_13.pdf

http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf

DNA ISOLATION AND PURIFICATION

DNA ISOLATION AND PURIFICATION

DNA isolation methods

Many different methods and technologies are available for the isolation of genomic DNA. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA. Removal Of proteins is typically achieved by digestion with proteinase K, followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology). DNA is usually recovered by precipitation using ethanol or isopropanol. The choice of a method depends on many factors: the required quantity and molecular weight of the DNA, the purity required for downstream applications, and the time and expense. Several of the most commonly used methods are detailed below, although many different methods and variations on these methods exist. Home-made methods often work well for researchers who have developed and regularly use them. However, they usually lack standardization and therefore yields and quality are not always reproducible. Reproducibility is also affected when the method is used by different researchers, or with different sample types.

The separation of DNA from cellular components can be divided into four stages:

1. Disruption

2. Lysis

Cell walls and membranes must be broken to release the DNA and other intracellular components. This is usually accomplished with an appropriate combination of enzymes to digest the cell wall (usually lysozyme) and detergents to disrupt membranes. We use the ionic detergent Sodium Dodecyl Sulfate (SDS) at 80˚C to lyse E. coli.

3. Removal of proteins and contaminants

RNA is usually degraded by the addition of RNase. The resulting oligoribinucleotides are separated from the high molecular weight (HMW) DNA by exploiting their differential solubilities in non-polar solvents (usually alcohol/water).

Proteins are subjected to chemical denaturation and/or enzymatic degradadtion. The most common technique of protein removal involves denaturation and extraction into an organic phase consisting of phenol and chloroform.

Another widely used purification technique is to band the DNA in a CsCl density gradient using ultracentrifugation.

4. Recovery of DNA

In some methods, stages 1 and 2 are combined.

Reference:

www1.qiagen.com/literature/brochures/Gen_DNA_Pur/1019469_BROS_DNYTi_p10_13.pdf

http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf