LAB 6: BACTERIAL DNA EXTRACTION

Introduction

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. The information in DNA is stored as a code made up of four chemical bases which are adenine (A), guanine (G), cytosine (C), and thymine (T). The order of these bases determines the information available for building and maintaining an organism. The DNA of most bacteria is contained in a single circular molecule, called the bacterial chromosome. In addition to the chromosome, bacteria often contain plasmids which is a small circular DNA molecules. Plasmids are usually used to purify a specific sequence since they can be easily be purified away from the rest of the genome. So, plasmids need to be isolated for their use as vectors and also for molecular cloning. 

There are two large groups of bacteria based on their different cell wall constituents, which are gram-positive and gram-negative bacteria. Gram-positive bacteria are bacteria that give a positive result in the Gram stain test. They appear to be purple colored when seen through a microscope due to the thick peptidoglycan layer in the bacterial cell wall which retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test. Examples are Lactobacillus fermentum, Bacillus subtilis, Streptococcus agalactiae. Gram-negative bacteria cannot retain the violet stain after the decolorization step; alcohol used in this stage degrades the outer membrane of gram-negative cells making the cell wall more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take up the counter strain and appear as red or pink. Examples are Escherichia coli and Salmonella typhimurium .

DNA extraction is a routine procedure used to isolate DNA from the nucleus of cells. Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids. The reason for this procedure is to separate the plasmid DNA from its associated proteins so that further manipulations can be done to it. DNA extraction involves several processes such as lysozyme treatment, centrifugation, protein denaturation, homogenization etc. The lysozyme treatment is important to break down bacterial cell walls to improve nucleic acid extraction efficiency.

The GF-1 Bacterial DNA Extraction Kit is designed for rapid and efficient purification up to 20μg of a high molecular weight genomic DNA from 1-3 ml either Gram-negative or Gram–positive bacteria. This bacterial purification kit applies the principle of a minicolumn spin technology and the use of optimized buffers to ensure that only DNA is isolated while cellular proteins, metabolites, salts and other low molecular weight impurities are removed during the subsequent washing steps.

Figure 1: GF-1 Bacterial DNA Extraction Kit

DNA concentration and purity can be assessed using absorbance (optical density) of DNA solution. The intensity of absorbance of the DNA solution is measured at wavelengths 260 nm ad 280 nm. DNA absorb ultraviolet (UV) light due to the heterocyclic rings of the nucleotides; the sugar-phosphate backbone does not contribute to absorption. To ensure the concentration reading is accurate, the absorbance reading should be within the linear range of the spectrophotometer. The Lambert-Beer law relates the absorption of light to the properties of the material through which the light is travelling. Several factors that can influence the accuracy of A260/A280 ratio includes type of protein present, absorbance of phenol and other contaminants at 280 nm and pH of DNA solution.

Objective

- To understand and practice proper technique of isolation process of plasmid DNA from bacteria.

- To determine the DNA concentration using Beer-Lambert equation

Materials and Apparatus

Columns 

Collection tubes 

Receiver

Pipette

Incubator (Digital Dry Bath)

Centrifuge

Spectrophotometer

Microcentrifuge tube

Gram-negative bacteria strain, Escherichia coli

Gram-positive bacteria strain, Lactobacillus fermentum
Resuspension Buffer 1
Resuspension Buffer 2

Lysozyme
Proteinase K solution 

Ethanol
Wash Buffer 
Elution Buffer 

Procedure:

Reminder:

* All steps are to be carried out at room temperature unless stated otherwise.

* Wash buffer (concentrate) has to be diluted with absolute ethanol before use.

* Pre-heat Elution Buffer to 65°C (optional).

1. Centrifugation. 1ml of bacteria culture grown overnight or culture grown to log phase is turned into pellet by centrifugation at 6000 x g for 2 minutes at room temperature. The supernatant is decanted completely.

2. Resuspension of pellet. 100µl Buffer R1 is added to the pellet and the cells are resuspended completely by pipetting up and down.

3. Lysozyme treatment. For Gram-negative bacteria strain (Escherichia coli), 10 µl lysozyme (50mg/ml) is added into the cell suspension. For Gram-positive bacteria strain (Lactobacillus fermentum), 20 µl lysozyme (50mg/ml) is added into the cell suspension. They are mixed thoroughly and incubated at 37°C for 20 minutes.

4. Centrifugation. Digested cells are centrifuged at 10000 x g for 3 minutes and pellet is formed. The supernatant is decanted completely.

5. Protein denaturation. Pellet is resuspended in 180 µl of Buffer R2 and 20 µl of Proteinase K is added. It is mixed thoroughly. Then, it is incubated at 65 °C for 20 minutes with occasional mixing every 5 minutes.

6. Homogenization. 400 µl without RNase A treatment of Buffer BG is added and is mixed thoroughly by inverting tube several times until a homogenous solution is obtained. It is incubated for 10 minutes at 65°C.

7. Addition of ethanol. 200 µl of absolute ethanol is added, then is mixed immediately and thoroughly.

8. Loading to column. The sample is transferred into a column assembled in a clean collection tube. It is centrifuged at 10000 x g for 1 minute. Flow through is discarded.

9. Column washing. The column is washed with 650 µl of Wash Buffer and is centrifuged at 10000 x g for 1 minute. Flow through is discarded.

10. Column drying. The column is centrifuged at 10000 x g for 1 minute to remove residual ethanol.

11. DNA elution. The column is placed into a clean microcentrifuge tube. 50 µl of pre-heated Elution Buffer, TE buffer is added directly onto column membrane and is allowed to stand for 2 minutes. It is centrifuged at 10000 x g for 1 minute to elute DNA.

12. The DNA content is measured using spectrophotometer with absorbance at 260nm and 280nm.

Figure 2: Gram-positive bacteria (Lactobacillus fermentum) and Gram-negative bacteria (Escherichia coli).

Figure 3: Centrifugation.

Figure 4: Resuspension.

Figure 5: Incubation using digital dry bath.

Figure 6: DNA content is measured using spectrophotometer with absorbance at 260nm and 280nm.

Result

Table 1: A260/A280 ratio of the samples

Bacteria	Optical Density		Ratio (260/280)
	260	280
Escherichia Coli (Gram-negative)	0.069	0.035	1.97
Lactobacillus fermentum (Gram-positive)	0.264	0.157	1.681

The DNA concentration of the bacteria is calculated by using Beer-Lambert equation shown below:

 A=ƸCl

A= A260 (absorbance) Ƹ=0.020 (molar extinction coefficient) C= concentration of DNA l= 0.051 (light pathlength)

                                                  

For the DNA concentration of Escherichia Coli (Gram-negative),

Let C= DNA concentration of Escherichia Coli

By using Beer-Lambert equation,

A=ƸCl 0.069= 0.020 (C) (0.051) C=0.069/(0.020x0.051)  C= 67.64 µg/ml

For the DNA concentration of Lactobacillus fermentum (Gram-positive),

Let C= DNA concentration of Lactobacillus fermentum

By using Beer-Lambert equation,

A=ƸCl 0.264= 0.020 (C) (0.051) C=0.204/(0.020x0.051)  C= 258.82 µg/ml

Discussion

     Before discussing the result, a brief explanation on the Gram-positive bacteria, Gram-negative bacteria, lysozyme, precaution and others are discussed. Gram staining is a technique developed by Christian Gram in 1884 which is used to strain bacteria. The bacteria which retain the colour of the stain are called Gram-positive bacteria whereas the bacteria which lose the colour of the stain are called Gram-negative bacteria. Gram-positive cell is bacterial cell having wall composed of thick layer of peptidoglycan containing teichoic acids and retaining the crystal violet dye used in the Gram staining procedure, appearing purple when seen through a microscope. Gram-negative cell is bacterial cell having a wall composed of a thin layer of peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane, besides that it do not retain the crystal violet stain used in the Gram staining method.

    Lysozyme is an enzyme that destroy the bacterial cell walls by catalytic hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan.

        

     In this experiment, some precaution steps must be taken. For instance, when re-suspend the cell completely by pipetting up and down, place the pipette on the same position in order to make sure complete cell re-suspension. Besides, when centrifuge, place the column which has a triangle marks on the edge to ensure that the process achieves optimization.

       

   OD260 unit, is a spectrophotometric measurement of an oligonucleotide. It is a normalized unit of measurement that is defined as the amount of oligonucleotide required to give an absorbance reading of 1.0 at 260 nm in 1.0 mL of solution using a 1 cm light path. Each of the bases in a nucleic acid strand has an absorbance at or near 260 nanometers, due to their conjugated double bond systems. Because the exact base sequence and composition is known, the OD260 unit is a precise method to quantify an oligonucleotide. Utilizing absorbance measurements is the recommended method for quantitating or aliquotting an oligonucleotide. Absorption at 280 nm indicates the presence of proteins due to the strong absorption by the aromatic amino acids.

           

     Ratio between the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of the DNA. A ratio of approximately in the range of 1.7-1.9 is generally accepted as “pure” for DNA. If the ratio is lower than this range, the DNA cannot be considering as “pure”. There are few factors that cause the low DNA ratio reading, one of that is caused by protein or phenol contamination that absorb strongly at or near 280 nm. Moreover, other factors include incomplete cell suspension, low elution efficiency, column clogged and incomplete protein denaturation.

            

     From the result obtained, the ratio of A260 to A280 for Escherichia Coli is 1.97. This indicates the ratio of DNA in the extract is not high enough to be considered as “pure”. This value is slightly higher than the range of 1.7 to 1.9. This deviation may due to the contamination by the presence of RNA. Mild alkaline condition may also contribute to the increase in the ratio of A260/A280.

            

     The ratio of A260 to A280 for Lactobacillus fermentum is 1.68. The value calculated is slightly lower but very close to the range of 1.7 to 1.9 which means that the materials that are extracted are mainly DNA with little contamination by other materials like proteins.

           

     The concentration of DNA in E.coli obtained from the result is 67.64 µg/ml while the concentration of DNA in L.fermentum is 258.82 µg/ml. It is clearly shown that more DNA are extracted from L.fermentum. This may be caused by the technical errors done in the procedure of extracting the DNA from E.coli, resulting in lower level of DNA extracted. We can also compare the readings of DNA concentration of each bacteria with the ratios of OD260/OD280 respectively. For E.coli, the ratio obtained is 0.07 higher than the range where “pure” DNA is extracted. On the other hand, for L.fermentum, the ratio is only 0.02 lower than the range. Thus, the concentration of DNA extracted is higher in L.fermentum than E.coli.

Conclusion

DNA extraction requires a proper method and technique in order to obtain high DNA concentration. The bacterial extract containing DNA is placed in a spectrophotometer to measure the absorbance using light wavelengths at 260 nm and 280 nm. The ratio of A260/A280 gives the estimate of the DNA purity in the extract. The best ratio that contains highest DNA purity is between 1.7 to 1.9. Then, Beer-Lambert equation is used to calculate the concentration of DNA.

Reference

https://www.promega.com/-/media/files/resources/application-notes/pathlength/calculating-nucleic-acid-or-protein-concentration-using-the-glomax-multi-microplate-instrument.pdf?la=en

http://www.ogt.com/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality