Laboratory 10: DNA and Molecular Biology

DNA isolation

1. Homogenization
2. Deproteinization
3. Precipitation

homogenization

the process of separating plant cell walls, plasma membranes and nuclear membranes to release the DNA. This is usually performed with a blender or mortar and pestle. Detergents are added to the homogenizing medium to help solubilize the cells’ membranes and denature the proteins

deproteinization

involves the stripping of chromosomal proteins from the DNA. The proteins can then be denatured and precipitated from the homogenate containing the DNA

precipitation

occurs when ice-cold ethanol is added to the homogenate and the DNA separates at the interface between the alcohol and the homogenate containing all the plant tissue components

Isolation of DNA from Strawberries

1\. Measure out 100 ml of strawberries and place them into a blender. Add 200 ml of cold water and 1/2 tsp of salt to the blender.

2\. Homogenize the mixture at the highest speed for 15 to 20 sec. Homogenization breaks open the cells and releases their contents (carbohydrates and proteins. The homogenate should look like a thick soup.

3\. Pour your strawberry-cell soup through a cheese cloth to separate the large particulate from the homogenate. Collect the filtrate in a large beaker.

4\. Add 30 ml of detergent to the beaker and swirl the mixture for at least 5 minutes. Let the mixture stand for 5-10 minutes.

5\. Pour the mixture into test tubes until they are about 1/3 full. Add a pinch of enzyme (Adolph's Meat Tenderizer) to each test tube and stir gently. Gently stir the mixture; too much agitation will disrupt the DNA.

6\. Tilt each test tube and slowly pour alcohol (70-95% isopropyl or ethanol) down the side of the tube. Pour enough alcohol so that the volume equals the volume of the strawberry mixture. An alcohol layer will form at the top of the strawberry mixture. DNA from the strawberry soup will rise into the alcohol layer.

7\. Use a wooden stick to gently spool out the DNA from the tube. The stringy slightly gelatinous material that attached to the stick is DNA Note: If the DNA has been damaged, it will still precipitate, but as white flakes that cannot be collected on the stick. If you cannot see any DNA allow the mixture with alcohol to sit for 30 minutes, the DNA should precipitate into the alcohol layer

Feulgens stain

staining technique used to visualize DNA

DNase

a group of enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA

DNA fingerprinting

a commonly used technique for identifying the source of DNA; powerful evidence in paternity cases and has been used successfully to identify the bodily remains of victims, when visual evidence is absent

hair, saliva, semen, and blood

common sources of DNA for extraction

Restriction fragment length polymorphisms (RFLPs)

once DNA is isolated, the large nucleic acid chains must be digested into these smaller fragments using restriction enzymes

restriction enzymes

cut DNA at very specific sequences of bases; they will digest the DNA everywhere the recognition sites appear on the strand, leaving behind smaller DNA fragments which can enter an agarose gel when an electrical current is applied

recognition sites

the site recognized by a restriction enzyme to cleave DNA

polymerase chain reaction (PCR)

a valuable technique commonly used in molecular biology labs, allowing scientists to take a very small amount of isolated DNA and amplify it several orders of magnitude, producing millions of copies of the desired DNA

PCR Steps

1. DNA of interest must be extracted from the appropriate tissue
2. Synthetic primers (oligonucleotides) are constructed which correspond to the ends of the DNA
3. These primer pairs, also called forward and reverse primers, bind to one of the two DNA strands
4. The DNA amplification process begins at the primers
5. Extra concentrations of primer are added to allow for the priming of the additional DNA as the amplification process continues
6. The PCR reaction tube will contain the extracted DNA, 4 deoxynucleotide triphosphates in buffer, and Taq DNA polymerase
7. The PCR mixture will be exposed to the following 3-step temperature cycle:


1. Denaturation
2. Annealing
3. Extension
8. This process will be repeated 20-50 times to amplify the amount of DNA produced
9. The PCR reaction takes place in a thermal cycler (PCR machine) pre-programmed to alternate to these temperatures
10. The PCR mix is then electrophoresed on a horizontal agarose gel

synthetic primers/oligonucleotide

consists of a short sequence of nucleotides that are synthesized to match a specific region of DNA; about 20-45 nucleotides long

Taq DNA polymerase

enzyme that has a high tolerance for heat and is able to withstand the temperatures of the PCR reaction

PCR reaction tube

contains extracted DNA, four deoxynucleotide (A, C, G, T) triphosphates in buffer, and Taq DNA polymerase

Denaturation

temperature of 94° C to melt the hydrogen bonds between the two strands of DNA

Annealing

temperature was decreased to 42° and 60° to hybridize the 2 primers on the DNA strands

Extension

temperature was then increased to 72° C (optimum temp for Taq DNA polymerase) allowing it to synthesize the new DNA strand

thermal cycler (PCR machine)

the PCR reaction takes place in this, which is preprogrammed to alternate to these temperatures

alternative splicing

this occurs when mRNA exons are assembled together in a different order from the original pre-mRNA transcript

autoradiography

used by scientists in the past to decipher a DNA sequence

autoradiography process

1. This method involved radioactive 35S-labelled nucleotides and polyacrylamide gels to separate the A, C, G, T nucleotides into different lanes
2. The radioactive gels were then incubated with a sheet of x-ray film and a visible picture of the DNA sequence formed on the film
3. Before the start of this experiment, double stranded DNA was denatured into individual single strands
4. Aliquots of the single stranded DNA were then placed into four tubes labeled A, C, G, and T
5. Nucleotide terminators were added to each of the four tubes and produced DNA fragments of varying lengths
6. The fragments were then electrophoresed on a vertical polyacrylamide gel
7. After the run was complete, the gel was dried and incubated with photographic film for a period of time and an audiograph developed

nucleotide terminators

stopped the growing DNA chain from making more A, C, G, and T nucleotides and produced DNA fragments of varying lengths

ligase

an enzyme which brings about the joining of two DNA strands or other molecules by a phosphate ester linkage of DNA

bacterial plasmid

a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules