After sample preparation, your target molecule will most likely require analysis. If your sample volume is too small, then amplification (via PCR or molecular cloning) is necessary to allow accurate downstream evaluation. Additionally, separation by electrophoresis can your identify specific species or fragments within your sample.
Amplification - Polymerase Chain Reaction (PCR)
One of the classic laboratory techniques in all of biology, ranging from ecology and evolution to biotechnology, is the polymerase chain reaction. PCR is an in vitro method for the amplification of a specific DNA template to quickly produce a large number of copies. Initially developed in 1983 by Kary Mullis; PCR helped replace the slower cloning techniques which required days of lab work.
PCR Reaction Components:
1. DNA or RNA template: which we want to amplify.
2. Nucleotides (dCTP, dGTP, dATP, dGTP): DNA and RNA building blocks, required for elongation
3. Primers: short nucleotide strands which anneal to the positions that we want to start and end the amplification.
4. DNA polymerase: the enzyme catalyzing the reaction.
5. Accessory additives like buffers and Mg2+
1. Denaturation: DNA is heated in a thermocycler to split the double-stranded molecule into two single strands.
2. Hybridization: Primers can anneal to their target sequences on the single-stranded DNA, during a cool-down phase.
3. Elongation: The single strands of DNA are extended via polymerases, which start from the primer sequences, resulting in two double-stranded DNA molecules.
4. The above three steps repeat until there is a lack of substrate or the product accumulation halts the reaction. After every cycle, the amount of product increases exponentially.
PCR was initially established to detect particular DNA sequences within a sample, a technique still commonly used today. However, many other applications have been elaborated over the past few decades. Nowadays different types of PCR exist:
· Real Time (qPCR) (quantitative)
· Reverse Transcription (RT-PCR)
Other variations include multiplex, digital and nested PCR. Additionally, adapted polymerases used in PCR may incorporate features such as a hot start mechanism or proofreading activity. Not only is PCR a key method for DNA replication, but it is also often used in cloning processes.
Amplification - Molecular Cloning
Despite the name, cloning is the process of copying either segments or the entire genome by utilizing the cell’s natural self-replication steps; simply, cloning refers to the procedure of making multiple copies of recombinant DNA. These molecular cloning methods allow for scientists to build a bank of DNA to work from, which are commonly referred to as DNA libraries.
In sum, molecular cloning steps typically involve the choice of a host cell and cloning vector, preparation of vector and target DNA, formation of recombinant DNA, transfection of recombinant DNA into a host cell, then selection and screening of host cells for target DNA.
Molecular cloning is similar to PCR, but rather than replicate DNA in vitro; cloning occurs within a living cell. However, molecular cloning can sometimes utilize PCR for preparing DNA that requires cloning.
Separation - Electrophoresis
The final stages of PCR and some methods of nucleic acid purification require electrophoresis; a common technique employed in laboratories to filter molecules based on size. In principle, electrophoresis separates particles based on electrical charge.
For example, DNA molecules are negatively charged. So, to separate them based on size, a positive electrical current is passed through a gel; forcing the DNA molecules to travel through the gel at a velocity inversely proportional to their size, depending on the gel porosity. So larger molecules will have a more difficult time to pass through the gel when compared to smaller molecules, thus allowing for separation between DNA molecules.