Protocol for Real-Time PCR

This protocol describes the detailed experimental procedure for real-time RT-PCR using SYBR Green as was mentioned in Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research 31(24): e154; pp.1-8. Please refer to this paper and the PrimerBank Help page for more background information. The procedure begins with reverse transcription of total RNA. The cDNA is then used as template for real-time PCR with gene specific primers. You may need to modify this protocol if you use different reagents or instruments for real-time PCR.

Time required

	cDNA synthesis: 2 hours.
	real-time PCR: 2 hours.
	Dissociation curve analysis: 0.5 hour.

DNA Precipitation

Overview

This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in. It can also be coupled with phenol chloroform extraction for the purifying nucleic acids. This protocol also works for RNA precipitation (take care to use RNAse free materials in this case).

Materials

• 3M NaOAc pH 5.2
• EtOH 95%
• Glycogen (optional)

DNA Precipitation protocol (2)

Solutions/reagents:

• 3M Sodium Acetate solution
• Glycogen
• 95% EtOH
• 70% EtOH
• water
• buffer
• DNA sample

Equipment:

• Centrifuge

Steps:

1. Measure out 0.1 volume 3M Sodium Acetate solution into DNA sample.
2. Measure out 1 µl of Glycogen into Eppendorf tube (1).
3. Add 2 volumes 95% EtOH.
4. Option 1: Store at -20°C for 12 hrs(overnight).
   (or)
   Option 2: Store at -80°C for 30 mins.
5. Centrifuge at maximum speed for at least 15 mins at room temperature, gently aspirate out the supernatant and discard it.
(Optional)
Add 1 ml of 70% EtOH.
Store at room temperature for 5 mins.
(Optional)
Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.

6. Dry the pellet in air for 10 - 15 mins.
   Dry until all the liquid is gone.
7. Option 1: Add water to pellet.
   (or)
   Option 2: Add buffer to water.
   Resuspend pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 25 mins

LAMP - Loop Mediated Isothermal Amplification

LAMP - Loop Mediated Isothermal Amplification
"LAMP" stands for Loop-mediated Isothermal Amplification. This technology was developed by Notomi et al. It is a very sensitive, easy and time efficient method. The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.

Types of Primers used in LAMP

LAMP is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions of the target gene. The four primers used are as follows:
1. Forward Inner Primer (FIP): The FIP consists of a F2 region at the 3'end and a F1c region at the 5'end. The F2 region is complementary to the F2c region of the template sequence. The F1c region is identical to the F1c region of the template sequence.
2. Forward Outer Primer (FOP): The FOP (also called F3 Primer) consists of a F3 region which is complementary to the F3c region of the template sequence. This primer is shorter in length and lower in concentration than FIP.
3. Backward Inner Primer (BIP): The BIP consists of a B2 region at the 3'end and a B1c region at the 5'end. The B2 region is complementary to the B2c region of the template sequence. The B1c region is identical to the B1c region of the template sequence.
4. Backward Outer Primer (BOP): The BOP (also called B3 Primer) consists of a B3 region which is complementary to the B3c region of the template sequence.

Multiplex PCR

Introduction of Multiplex PCR

Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

Types of Multiplex PCR

Multiplexing reactions can be broadly divided in two categories:

1. Single Template PCR Reaction
This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template.
2. Multiple Template PCR Reaction
It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.

NASBA Technology

Introduction to NASBA

RNA detection is commonly done using RT-PCR, a time consuming process often resulting in false positives due to cross contamination. Alternatively, nucleic acid sequence-based amplification (NASBA) is a one step isothermal process for amplifying RNA. NASBA has proven to be successful in detection of both viral and bacterial RNA in clinical samples.
A NASBA reaction consists of avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase and RNase H with two oligonucleotide primers. The amplification is more than 1012 fold in 90 to 120 minutes. The amplification of ssRNA is possible only when the denaturation of dsDNA does not occur. As NASBA is an isothermal process, it is thus possible. The NASBA reaction does not get false positives caused by genomic dsDNA, as in the case of RT-PCR.

PCR Primer Design Guidelines

PCR (Polymerase Chain Reaction)

Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.

PCR involves the following three steps: Denaturation, Annealing and Extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94^oC, 60^oC and 70^oC respectively. Good primer design is essential for successful reactions. 

Polymerase Chain Reaction (PCR)

Introduction:

            The molecular technique called PCR is in vitro amplification of a specific segment of DNA using a thermostable enzyme.  Although it is a fairly new technique, invented in 1985 by Cary Mullis, it is widely used in hundreds of labs all over the world.  The PCR process makes millions of copies of DNA in just a few hours.  It is a replication reaction, which uses reagents very similar to what is needed for DNA replication inside a cell.  Each strand serves as a template for synthesis of its complementary strand.

            PCR has many applications. 1) It is a form of direct cloning of DNA (without the need for bacteria).  This is convenient when there is little DNA to work with.  2) PCR can produce a DNA fingerprinting pattern for forensics purposes, such as identifying blood at a crime scene.  3) PCR can be used in prenatal diagnosis of genetic diseases. 4) It can be used for evolutionary analyses, to look at genetic relationships among or within taxa (genera, species, populations).  5) PCR can detect allelic sequence variation and chromosomal rearrangements.  6) It is involved in the DNA sequencing process.  7) It is the newest technique in detecting viral or bacterial infection within a host. 

Gel Electrophoresis

Introduction:

The size of a DNA fragment can be estimated by gel electrophoresis.  This technique separates fragments by charge, size (molecular weight) and shape.  First, an agarose gel is made with slots (wells) in it.  The DNA sample is dispensed in the well, a buffer solution is placed in the apparatus, and an electric current is run through the gel.   DNA molecules are negatively charged due to the phosphates in its backbone, and when placed in an electric field starting at the negative (black) electrode, it will migrate towards the positive (red) electrode.  The gel is a complex molecular network containing narrow passages.  Smaller DNA molecules pass through more easily (less friction) and migrate faster through the gel than larger size fragments.   Linear molecules also migrate faster through a gel, compared to globular  forms.  

            Since the DNA fragments generated in this experiment are all negatively charged and linear in shape, the only variable to observe during electrophoresis is size.  To determine the size of a molecule, a standard or positive control is run concurrently in the gel.  The standard consists of fragments of DNA of known size.  A positive control contains the same size fragment (in our case 662 bp) as potential positive samples.