Common techniques in DNA analysis

In a nutshell

The polymerase chain reaction, electrophoresis and restriction enzymes are important to understand as they are vital for methods such as DNA profiling, DNA sequencing and genetic engineering. This summary discusses these common techniques.

Polymerase chain reaction

The polymerase chain reaction (PCR) is a technique developed in $1983$ by Kary Mullis to amplify a section of DNA.

Biology; Manipulating genomes; KS5 Year 12; Common techniques in DNA analysis — A: Denaturation, 1. DNA fragment, 2. Separated strands.
B: Primer annealing, 3. DNA primer, 4. DNA with primers attached.
C: Elongation, 5. Nucleotides, 6. DNA polymerase (white) attaches the nucleotides, 7. New DNA fragments

1.	The DNA sample is mixed with free DNA nucleotides, primers and a DNA polymerase like Taq DNA polymerase. Primers are short sequences of bases that are complementary to the bases at the start of your fragment of interest. *Curiosity:* Taq polymerase originates from the thermophilic bacterium Thermophilus aquaticus and it is used because it can withstand the high temperatures that it experiences in PCR.
2.	The DNA must then be denatured to break the hydrogen bonds between the complementary base pairs in the double-stranded DNA. The solution is therefore heated to $95\ \degree C$ .
3.	The solution is then cooled to $50-65\ \degree C$ to allow the primers to anneal (attach) to the single strand. This creates a small section of double-stranded DNA at the end of each single-strand.
4.	Taq polymerase will bind to the short section of double-stranded DNA. The solution is heated to $72\ \degree C$ as this is its optimum temperature. The enzyme catalyses the reaction that joins complementary free nucleotides to the single-stranded DNA molecule. This extends the DNA molecule. *Note:* The DNA polymerase always starts at the end of the strand with the primer and travels from the $5'$ end to the $3'$ end of the strand.

At the end of the first PCR cycle, there will be two new strands produced. The cycle then repeats a maximum of $40$ times.

Electrophoresis

Electrophoresis is a technique that applies an electrical current to a sample of DNA in order to separate out fragments by their size.

Biology; Manipulating genomes; KS5 Year 12; Common techniques in DNA analysis — A: Filling the wells, 1. Pipette, 2. Well, 3. Agarose gel in the electrophoresis tank that is filled with buffer solution.

B: The electrophoresis tank, 4. Power supply, 5. Cathode, 6. Anode, 7. Direction of fragment migration, 8. Gel in the tank covered with buffer solution.

C. Electrophoresis gel showing the separated fragments.

Preparation

1.	Agarose gel is commonly used for electrophoresis. It will be a solidified gel with a row of wells at one end.
2.	The slab of gel must be placed into an electrophoresis tank. The end of the gel with the wells must be placed closest to the negative electrode.
3.	Buffer solution is added to the electrophoresis tank. The buffer allows the current to travel through the gel whilst maintaining the pH.

Loading samples

1.	Add loading dye to your DNA sample. This will make the sample easier to see and ensure it sinks to the bottom of the well.
2.	Using a micropipette, add a set volume of the DNA sample to the well. Example $10\ \mu m$ of DNA sample.
3.	Repeat this process with each DNA sample. *Note:* Ensure you change the micropipette tip for each DNA sample.

Running the gel

1.	Secure the lid on the electrophoresis tank and connect the cables to a power supply.
2.	Set the power supply to a specified voltage and switch it on. *Note:* You should see small bubbles appearing at the cathode.
3.	The DNA fragments carry a negative charge, therefore they will migrate towards the anode. Smaller DNA fragments will move faster and therefore they will appear lower on the gel.
4.	Stop the current after approximately $30\ minutes$ .
5.	Remove the gel from the tank and apply a stain solution. Rinse the gel with water and observe the bands.

Note: The size of the fragments is commonly measured in $kilobases$ ( $kb$ ). This means that $1000\ bases = 1kb$ .

Protein electrophoresis

A similar method can be applied to separate proteins based on their size. However, proteins can be positively or negatively charged. Therefore, they are denatured in order to make them all negatively charged. They can then be separated by electrophoresis.

Restriction enzymes

Restriction enzymes can be used in order to create DNA fragments. DNA can sometimes have palindromic sequences.

Example

The sequence below would be read the same in opposite directions.

$5'-CAAGTACCG-3'\\3'-GCCATGAAC-5'$

Restriction enzymes recognise these palindromic sequences and cut the DNA when they arise. Different restriction enzymes will have different sized active sites that fit different recognition sequences.

Example

EcoRI has the recognition site of GAATTC and HindIII has the recognition site of AAGCTT.

A DNA sample can be incubated with a restriction enzyme. The enzyme will cut a DNA fragment out through a hydrolysis reaction.

Certain restriction enzymes will leave short tails of unpaired bases called 'sticky ends'. These ends can bind to a complementary sequence, allowing the DNA fragment to be inserted elsewhere.

Biology; Manipulating genomes; KS5 Year 12; Common techniques in DNA analysis — A. Blunt end, B. Sticky end