The Difference of the Genomic DNA Extraction Between Animal & Plant Lecture 3 – 02.11.2017 – FALL 2017 by Jasmin SUTJOVIC

The structure of double-stranded DNA is universal in all living cells, but differences occur in the methods for extracting genomic DNA from animal and plant cells. Genomic DNA is found in the nucleus of cells. The amount and purity of extracted DNA depends on the type and size of the cell, as certain cells contain more DNA and impurities than others.  

GENERAL DNA EXTRACTION Plant and animal cells treated with a soapy substance will degrade the lipids in the cell and nuclear membranes. The DNA mixture will then separate from the cell membranes and proteins. The DNA in solution can be precipitated using alcohol. Depending on the amount in the sample, DNA may be visible by the naked eye. Such a simple procedure does not necessarily produce DNA of high purity

PLANT AND ANIMAL CELLS Plant cells are distinguishable from animal cells by their rigid cell wall and organelles like the chloroplast. They also contain proteins and enzymes that play a role in photosynthesis. Some plant cells are polyploidy, meaning they have more than one copy of each chromosome per cell. Cellular processes occurring in plants such as photosynthesis produce a range of secondary metabolites. Animal cells do not have a cell wall, but still need to be treated with chemicals like sodium dodecyl sulphate (SDS) to disrupt the cell membrane to release genomic DNA.

PLANT DNA EXTRACTION Plant genomic DNA is more difficult to extract because of the plant’s cell wall, which is removed by homogenization, or by adding cellulase to degrade the cellulose that makes up the cell wall. Also, the metabolites present in the plant cell may interfere with genomic DNA extraction by contaminating the DNA sample during the precipitation process.

ANIMAL DNA EXTRACTION Peripheral blood leukocytes are a main source of animal genomic DNA, but sample collection is difficult as blood must be withdrawn from the animal. Blood contains a range of compounds like proteins, lipids, white blood cells, red blood cells, platelets, and plasma, which can contaminate the DNA sample. The primary contaminant of animal DNA extracted from blood samples is heme, the non-protein component of hemoglobin.

DIFFERENCES The differences between plant and animal DNA lie in the sequence of bases in the helix. Compounds found in plant cells are absent in animal cells, and DNA base sequences reflect this, as the genomic plant DNA is often larger than animal DNA. These differences affect extraction methods, as it impacts on yield and purity of DNA.

The C1V1 = C2V2 Equation Explained Lecture 3b

The equation has four components: C1 = Initial concentration of solution V1 = Initial volume of solution C2 = Final concentration of solution V2 = Final volume of solution

Put together, the equation translates to: the starting concentration multiplied by the starting volume is equal to the final concentration multiplied by the final volume. Basically, if you have three of the four components of the equation then you can use these within the formula to calculate the unknown component. All you have to do is to rearrange the formula for your needs.

For example, if you want to calculate the final volume of a solution you would change the formula to: Or, if you want to calculate the initial starting concentration of a solution you would use:

Calculate the amount of 10 μM forward primer solution to add to a PCR reaction (25 μL total reaction) to make a final concentration of 0.4 μM forward primer in the reaction. Next, we need to fill in what we know. We know the values for C2 (0.4), V2 (25) and C1(10). So: V1 = (0.4 x 25) / 10 V1 = 10 / 10 V1 = 1 Therefore, in this example, we would need to add 1 μL of 10 μM forward primer solution to a PCR reaction containing a total volume of 25 μL to achieve a final forward primer concentration of 0.4 μM.

Calculate the amount of water you need to add to make a final concentration of 70% ethanol solution by using 100 mL of pure (100%) ethanol. In this example, we are asked to calculate the final volume (V2). Therefore, the equation will look like: We know the starting concentration (C1) of pure ethanol is 100%, the volume (V1) of pure ethanol we have is 100 mL and the final concentration (C2) we want to make is 70%. Putting this into the equation will look like: V2 = (100 x 100) / 70 V2 = 10,000 / 70 V2 = 142.9 The final volume we need to make therefore is 142.9 mL. We know 100 mL of that is the 100% pure ethanol, so the volume of water must be 42.9 mL (142.9 – 100 = 42.9). So, adding 42.9 mL of water to 100 mL of 100% pure ethanol will achieve a final concentration of 70% ethanol.

DNA quantification methods Lecture 4 09.11.2017 – FALL 2017 DNA quantification methods

Introduction DNA yield can be assessed using various methods including absorbance (optical density), agarose gel electrophoresis, or use of fluorescent DNA-binding dyes. All three methods are convenient, but have varying requirements in terms of equipment needed, ease of use, and calculations to consider.

1. Absorbance Method We need a spectrophotometer equipped with a UV lamp, UV-transparent cuvettes (depending on the instrument) and a solution of purified DNA. Absorbance readings are performed at 260nm (A260) where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution. To ensure the numbers are useful, the A260 reading should be within the instrument's linear range (generally 0.1–1.0).

Animation https://media.giphy.com/media/7DWHASPMPtoQ0/source.gif

Double beam spectrophotometer system

DNA yield (µg) = DNA concentration × total sample volume (ml) How we measure? DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml DNA yield (µg) = DNA concentration × total sample volume (ml)

What about RNA? However, DNA is not the only molecule that can absorb UV light at 260nm. Since RNA also has a great absorbance at 260nm, and the aromatic amino acids present in protein absorb at 280nm, both contaminants, if present in the DNA solution, will contribute to the total measurement at 260nm. Additionally, the presence of guanidine will lead to higher 260nm absorbance. This means that if the A260 number is used for calculation of yield, the DNA quantity may be overestimated. Guanidine , strong base , a functional group of arginine amino acid

DNA purity DNA purity (A260/A280) = (A260 – A320 ) ÷ (A280 – A320 ) The most common purity calculation is the ratio of the absorbance at 260nm divided by the reading at 280nm. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not indicate that the DNA is unsuitable for any application, but lower ratios indicate more contaminants are present. The ratio can be calculated after correcting for turbidity (absorbance at 320nm). DNA purity (A260/A280) = (A260  – A320 ) ÷ (A280  – A320 )

2. Fluorescence Methods The widespread availability of fluorometers and fluorescent DNA-binding dyes makes fluorescence measurement another popular option for determining of DNA yield and concentration. Fluorescence methods are more sensitive than absorbance, particularly for low-concentration samples, and the use of DNA- binding dyes allows more specific measurement of DNA than spectrophotometric methods allows. Hoechst bisbenzimidazole dyes, PicoGreen® and QuantiFluor™ dsDNA dyes selectively bind double-stranded DNA.

Materials required Materials required for fluorescence methods are: Fluorescent DNA binding dye Fluorometer to detect the dyes, and Appropriate DNA standards.

How it works? Fuorescence measurements are set at excitation and emission values that vary depending on the dye chosen. The concentration of unknown samples is calculated based on comparison to a standard curve generated from samples of known DNA concentration.  As with absorbance methods, dilution factor must be taken into account when calculating DNA concentration from fluorescence values.

3. Agarose gel electrophoresis To use this method, a horizontal or vertical gel electrophoresis tank with: an external power supply, analytical-grade agarose, an appropriate running buffer (e.g., 1X TAE) and an intercalating DNA dye along with appropriately sized DNA standards are required.

Loading and running the gel A sample of the isolated DNA is loaded into a well of the agarose gel and then exposed to an electric field. The negatively charged DNA backbone migrates toward the anode. Since small DNA fragments migrate faster, the DNA is separated by size. The percentage of agarose in the gel will determine what size range of DNA will be resolved with the greatest clarity

RNA or protein contamination??? Any RNA, nucleotides and protein in the sample migrate at different rates compared to the DNA so the band(s) containing the DNA will be distinct.

RNA contamination example

Protein contamination example

DNA degradation example

Our DNA isolation GEL Group 1

LAB: DNA quantification on qDROP GO Multiscan reader