PCR, Gel Electrophoresis, and Southern Blotting

By: D.D

What Is PCR?

• Stands for Polymerase Chain Reaction.

• A biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment or sequence of interest of DNA, via enzymatic replication, without using a living organism.

PCR Procedure (Steps 1 and 2)

• Mold samples are collected in the field with regular field sampling techniques.

• Samples can be air, dust or bulk.

• DNA is extracted from these samples by using a glass bead milling methodology to break down the cell walls of the mold spores.

PCR Procedure (Step 3)

• The DNA is then placed in a PCR "cocktail" mixture.

• This cocktail includes primers and probes designed specifically for the mold species that is to be identified and quantified.

• This mixture is contained in a tube that is placed in a thermal cycling/fluorescence detection instrument.

PCR Procedure (Steps 4)

• As the DNA is heated, the double helix splits apart, creating two template DNA strands.

PCR Procedure (Step 5)• During the annealing stage,

the sample cools. • Two single strand primers

find exact complementary locations to attach to single strand DNA templates.

• Additionally, a probe containing a fluorescent reporter at one end, and a quencher (to block the fluorescence) at the other end, attach to a specific location that is unique to the species of mold being analyzed.

PCR Procedure (Step 6)• The final state of the

PCR cycle is the extension stage.

• During this stage, complementary bases are added to the single strand DNA template.

PCR Procedure (Steps 7 and 8)

• This cycle of heating and cooling is repeated 40 times. As the cycles continue, the quantity of target DNA is replicated and increases exponentially, as does the fluorescent signal given off by the probe.

• Comparing this fluorescent signal to a standard, one is able to quantitate the amount of mold in the sample.

What Is Gel Electrophoresis?

• Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, and protein through an electric charge.

Uses of Gel Electrophoresis

• It is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

Gel Electrophoresis Procedure

• Step 1: The agarose gel with three slots/wells (S). • Step 2: Injection of DNA ladder into the first slot. • Step 3: DNA ladder injected. Injection of samples

into the second and third slot. • Step 4: A current is applied. The DNA moves

toward the positive anode due to the negative charges on its phosphate backbone.

Gel Electrophoresis Procedure (2)

• Step 5: Small DNA strands move fast, large DNA strands move slowly through the gel.

• Step 6: Add the color marker dye to the DNA ladder.

What Is Southern Blotting?

• A method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample.

• It combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization.

What Is Southern Blotting (cont.)

• The method is named after its inventor, the British biologist Edwin Southern. Other blotting methods, that employ similar principles, but using RNA or protein, have later been named in reference to Southern's name.

• As the technique was eponymous named, Southern blot should be capitalized, whereas northern and western blots should not.

Performing Southern Blotting

• 1. Digest the DNA with an appropriate restriction enzyme.

• 2. Run the digest on an agarose gel. • 3. Denature the DNA (usually while it is still

on the gel).• 4. Transfer the denatured DNA to the

membrane. • 5. Probe the membrane with labeled

ssDNA. This is also known as hybridization.6. Visualize your radioactively labeled target sequence.

Bibliography

• http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis#Procedure

• http://en.wikipedia.org/wiki/Gel_electrophoresis• http://en.wikipedia.org/wiki/Southern_blot• http://en.wikipedia.org/wiki/Polymerase_chain_re

action• http://forensica.com/_pcrproc.asp?x=pcr_proc.txt