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Introduction to Techniques

    Polymerase Chain Reaction
    • Standard Polymerase Chain Reaction (PCR)
    • Reverse Transcription PCR
    • Amplification Phases of PCR
    • Real-Time PCR
      • Gene Expression Detection
      • Allelic Discrimination

Nucleic Acid Analysis [Back to the top of this page]

Before embarking on very time consuming and at times expensive series of experiments, it is recommended that the quality and the concentration of RNA or DNA be evaluated. For total RNA quality assessment, investigators may choose to use the 2100 Bioanalyzer from Agilent technologies. This bioanalyzer is Agilent's highly successful microfluidics-based platform for the analysis of DNA, RNA, and proteins. As the first commercial, analytical instrument based on lab-on-a-chip technology the Agilent 2100 bioanalyzer has proven to be an excellent alternative to messy and labor-intensive gel electrophoresis techniques delivering fast, automated, high quality digital data. Click here for assay information.

Agilent Bioanalyzer 2100 ND-1000

The Nanodrop® ND-1000 available from the core lab is a full spectrum (220-750mm) spectrophotometer that measures 2ul of sample with high accuracy and reproducibility. For a rapid way to analyze DNA in a given sample, the BCL staff recommends Nanodrop technology. Applications include nucleic acid concentration and quality, fluorescent dye labeling density of nucleic acid microarray samples, purified protein analysis (A280), Bradford assay and BCA analysis of proteins, cell density measurements and general UV-Vis spectrophotometry. Utilizing a patented sample retention technology, surface tension alone holds the sample in place. This eliminates the need for cumbersome cuvettes. It is simple to use and has the capability to measure highly concentrated samples without dilution (75x higher concentration than samples measured by a standard cuvette spectrophotometer).

When precise nucleic acid measurement is paramount to the success of your experimental design, picogreen or ribogreen chemistries should be used to measure the amount and quality of nucleic acids contained in your samples.

Polymerase Chain Reaction[Back to the top of this page]

Standard Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a common method an investigator can use to rapidly create copies of specific fragments of DNA, starting from as low as a single copy.

There are three basic steps in PCR.

Unamplified DNA

1. Strand Denaturation

2.Primer Annealing

3. Primer Extension

First, the target genetic material must be denatured: The strands of its helix must be unwound and separated by heating to 90-96°C.

The second step is annealing of the primers to their complementary bases on the now single-stranded DNA template. Reducing the temperature below the melting temperature (50-60°C) of the primers allows for hybridization/annealing to occur.

The third is DNA synthesis also known as the extension step and generally takes place at 72°C. Starting from the primer, the enzyme Taq polymerase will copy the template strand using base pair complementarity (A pairs with T and C with G). Taq polymerase is an enzyme found in Thermophilus aquaticus, a bacterium that lives in extremely hot thermal vents, and is therefore able to withstand the intense heat of the PCR reaction without denaturing itself.

By cycling the temperature, the target DNA is repetitively denatured and copied. The process of denaturation, annealing and extension is repeated for an average of 25 to 30 cycles. During PCR a single copy of the target DNA, even if mixed in with other undesirable DNA, can be amplified to obtain billions of specific replicates called amplicons.

PCR in the core lab is usually performed on the ABI® 9700 thermocycler or the ABI® 2400 thermocycler. The 9700 series is equipped with Peltier heating/cooling technology, which allows for rapid cycling between temperatures. It is capable of handling up to 96, 200ul single tubes or one, 96-well plate.

In the core lab, stocks of five universal primers are available upon request. These include: M 13 forward and reverse, T3, T7 and SP6. We also offer custom oligonucleotide services through Integrated DNA Technologies (IDT). Please contact the BCL Assistant Director, Jennifer Holbrook for more information on designing and ordering custom primers.

When dealing with difficult templates, or when needing to optimize a PCR reaction, researchers can use the Stratagene RoboCycler® Gradient 96 that brings speed and innovation to the technique of PCR. Faster than conventional systems which ramp the temperature in a single block, the RoboCycler uses a robotic arm to quickly and efficiently move up to 96, 200ul tubes or one 96 well plate from one temperature block to another.

The unique 4-block design decreases cycling time up to 30% and achieves unmatched well-to-well temperature uniformity. Block #2 is a special brass gradient block that allows simultaneous evaluation of up to 12 different annealing temperatures. Investigators can change the temperature by 1 degree from column-to-column. This allows for quick optimization of the annealing reaction conditions in one easy experiment.

Reverse Transcription PCR

Reverse transcription is a process during which RNA molecules are transcribed into cDNA (copy-DNA) using an enzyme called reverse transcriptase (RT). cDNA can then be used as a template for PCR. RT-PCR is often used to identify which genes are transcribed in a specific tissue or in response to a certain treatment.

Routinely, investigators visualize the result of their PCR or RT-PCR by gel electrophoresis. This process uses an electrical current to help DNA fragments migrate through a gel-like polymer, such as agarose. The DNA molecules, which have an overall negative electrical charge, will migrate toward the anode, or positive electrode, and be separated by size. The larger fragments of DNA move through the sieves of the agarose gel more slowly than smaller DNA molecules. Usually, PCR products are visualized after gel electrophoresis by soaking the gel in a dye (ethidium bromide), which makes the DNA fluoresce under UV light.

Amplification Phases of PCR

The three phases of the PCR reaction are characterized by the reagent status present in the reaction tube.

In the early cycles, the reagents are in excess and the PCR is robust. This exponential phase is characterized by an exact doubling of the PCR product at each cycle. Thus, it is the most accurate place to measure PCR products.

During the linear phase, some of the reagent components are becoming limited, and the PCR efficiency is slowing down. This is a highly variable phase in the PCR reaction.

The last phase or plateau is achieved when reagents have been depleted and the reaction has stopped.

Under standard conditions, PCR products are generally visualized by agarose gel electrophoresis, when the reaction has reached the plateau phase.

For more accurate quantitation for gene expression analysis or gene/transgene copy numbers, using data collected during the exponential phase, Real-Time PCR or Real-time RT-PCR is available to the BCL investigators.

Real-Time PCR

Unlike conventional PCR, Real-Time PCR allows the investigator to monitor fluorescence produced by a reporter dye during each cycle. The amount of fluorescence is proportional to the amount of amplicons generated. This process allows for detection of all phases of amplification: Exponential, Linear and Plateau.

Using Real Time PCR, one can measure fluorescence while the reaction is still in the high precision exponential phase. The fewer cycles it takes to reach a detectable level of fluorescence, the greater the initial copy number of the targeted nucleic acid.

Real-time PCR has many applications including the precise measurement of changes in gene expression, overall gene expression quantitation, the detection of single nucleotide polymorphisms (SNPs) and post-PCR detection for allelic discrimination assays. It is also ideal for plus/minus assays to confirm the presence or absence of a pathogen, a transgene, or other specific target sequence.

Different chemistries are available to real time users depending on the application.

Gene Expression Detection

There are two sets of detection chemistries used to quantitate gene expression or transgene copy numbers currently in use in the core lab. The first one relies on the use of Syber Green and the second one is based on TaqMan probes. For more in-depth information about these detection chemistries please consult the core for additional booklet information.

Syber Green is a dye that interacts with double stranded DNA through binding of the nucleic acid major groove. It will fluoresce only when bound to the major groove of the DNA molecule. Syber Green does not fluoresce when in solution, however, it is important to note that this dye will bind to all double stranded DNA present in the sample (your specific PCR target, as well as other non-specific PCR amplicons present).

TaqMan probes on the other hand will allow for a more specific reaction. Probes consist of short oligo-nucleotides that are flanked by a reporter dye and a quencher dye.

The proximity of the Quencher (either when the probe is in solution or annealed to the target sequence) prevents fluorescence emission from the reporter dye. However, the reporter dye will fluoresce when cleaved away from the quencher. This event occurs during the PCR step of DNA synthesis due to the 5' nuclease activity of Taq polymerase. As the Taq DNA polymerase reaches the site where the TaqMan probe sits, the enzyme's nuclease activity will start cleavage of the probe, releasing the reporter dye from the quencher dye. This cleavage event is measured in real time by a CCD camera.

SABiosciences develops and markets a broad range of innovative and cost-effective research tools based on Syber Green technology. They provided researchers access to new tools for profiling gene expression focused on a biological pathway or disease state. Over 100 PCR Arrays are available covering Cytokines, Apoptosis, Cancer, Signal Transduction, and many other topics. They are also easily customized to fit any list of genes.

Allelic Discrimination

The primary source of genetic difference between any two humans is due to the presence of single nucleotide polymorphisms in their DNA. Single Nucleotide Polymorphisms (SNPs) are DNA variant/point mutations that are extremely useful as molecular genetics tools to follow inheritance of specific segments of DNA in linkage and association studies. Individuals that are homozygous for a SNP will harbor the same allele on both chromosomes. For example if the DNA variation is either a Cytosine (C) or Adenine (A) the homozygous individual genotype will either be C/C, or A/A. The most frequently encountered allele is often referred to as the major allele and the rare allele is called the minor allele. Individuals who have inherited two different alleles from their parents are heterozygous for a specific SNP. In the above example, one of the chromosome homologs contains the A allele while the other contains the C allele. The genotype of this heterozygous individual is A/C.

To use real time PCR as a tool to detect either of the two alleles of a SNP, one must use 2 TaqMan probes that will differ in sequence only at the site complementary to the SNP of interest. In the example below the first probe carries a Thymine (T) and will be able to bind to the allele harboring the Adenine (A) DNA variant. Note that this probe contains a fluorescent dye (VIC) that emits green fluorescence when cleaved away from the quencher. The second probe depicted below carries a Guanine (G), which will hybridize with the allele containing the complementary C base. Note that in this case the probe contains a fluorescent dye (FAM) that emits blue fluorescence when cleaved away from the quencher.

The probe that matches the SNP site is cleaved by the 5' nuclease activity of the Taq polymerase (as described in the gene expression section) thus releasing the SNP-specific dye. The mismatch probe is not able to anneal to the target, it remains intact and no fluorescence is emitted from this probe.

In instances when an individual is heterozygous at the SNP site (allele 1 and allele 2 as depicted in figure above) both probes can anneal with their specific target and thus both green and blue will be detected after cleavage of the probes during PCR.

The ABI PRISM® 7900 Sequence Detection System is a real-time PCR system that allows high throughput screening using a 384-well block.

To help investigators set-up routine high throughput 96 or 384 well formats, a robotics system is available in the core lab. The Biomek® 2000 Laboratory Automation Workstation is designed to meet the needs of investigators with simple, intelligent automation of liquid-handling tasks. Pipetting, diluting, and dispensing operations are performed quickly, easily and automatically. Volumes from 1ul to 200ul are pipetted with high precision. Multichannel tools are available to increase throughput.

Biomek 2000 ABI PRISM 7900

DNA Sequencing[Back to the top of this page]

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. Sequencing is performed by a DNA polymerase in buffered conditions, in the presence of a specific sequencing primer and nucleotide triphosphates (NTPs). Two sequencing methods are currently available from the BCL: Cycle Sequencing and Pyrosequencing.

Cycle Sequencing

Cycle sequencing is performed in the presence of a DNA template, a primer, an enzyme such as Taq polymerase, deoxynucleotides (dNTPs) and fluorescent dideoxynucleotides (ddNTPs). The ddNTPs are also referred to as "chain terminators" because once incorporated into the newly synthesized DNA chain no additional nucleotides can be added. The reaction mix is subjected to a cycle sequencing reaction performed in a thermocycler. During this reaction the sequencing reaction mix is heated to denature the double stranded DNA template. The primer is then able to hybridize to its complementary location on the template and polymerase can begin to elongate the primer. DNA denaturation is usually performed at 95°C, annealing at 50°C and synthesis at 60°C. Each time that the polymerase adds one of the ddNTPs in place of dNTPs the elongation of that particular fragment stops, leaving a dye-labeled nucleotide at the end. Because the event is random, fragments generated will be of length differing by 1 base. The result is many different sized fragments of the original template each labeled at the end with a dye that corresponds to the last base added. These dye-terminator labeled fragments can be separated by size by electrophoresis in a single capillary.

The ABI PRISM® 3130 xl -16 Capillary Genetic Analyzer is a high throughput automated system that is capable of determining base sequence for de novo and resequencing applications. Spectral detection of dye-labeled DNA fragments is performed, in which the final base at the end of each fragment is identified. This process recreates the original sequence of As, Ts, Cs, and Gs for each sample of DNA. The ABI DNA Sequencing Analysis software analyzes the resulting raw data, and the output is a four-color chromatogram showing peaks that represent each of the four DNA bases along with a text document of the sequence.


Pyrosequencing works by hybridizing a primer to a single stranded PCR amplified DNA template and incubating it with DNA polymerase, ATP sulfurylase, luciferase and apyrase in the presence of the substrates adenosine 5' phosphosulfate (APS) and luciferin. Then the first of four deoxynucleotide triphosphates (dNTP) is added. The primer elongates in the presence of DNA polymerase and each time a dNTP is incorporated onto the growing strand, a pyrophosphate is released. ATP sulfuylase converts the pyrophosphate to ATP in the presence of adenosine 5' phosphosulfate. The ATP catalyzes the conversion of luciferin to oxyluciferin, which generates visible light that is proportional to the amount of ATP generated.

The light is detected by a CCD camera and is represented by a peak in a pyrogramTM. Each light signal or peak is proportional to the number of nucleotides that were incorporated on the growing strand. Apyrase continually degrades unincorporated dNTPs and excess ATP to internally optimize the reaction. Another nucleotide can then be added when the degradation is complete, and the process repeated for all four nucleotides. The sequence can be determined from the signal peak in the pyrogramTM.

The Biotage PSQ 96MA pyrosequencer is currently available in the core lab for DNA sequencing of short segments and for rapid screening of SNPs.

The PSQ 96MA System uses Pyrosequencing technology (sequencing by synthesis) to enable real time detection of DNA sequencing events. Capabilities include automatic multiplex genotyping of single nucleotide polymorphisms (SNPs) and other mutations, allele frequency quantification and sequencing of short-strand DNA templates. Internal controls, which are provided by the sequence data, eliminate ambiguous results and the need to re-verify. Genetic variations can be analyzed with accuracy and confidence in a gel free operating environment. The results provide base sequence information, polymorphic position context and statistical analysis options.

DNA Fragment Analysis[Back to the top of this page]

DNA fragment analysis (here, shortened to FA) is a colloquially used term to generally describe the analysis or characterization of relatively short fragments of DNA (generally less than 10 kb, often less than 1.0 kb) generated by DNA polymerase-catalyzed reactions or by endonuclease digestion. Many types of methodologies and technology platforms are used to carry out FA, which is done for the most part to genetically characterize a genetic locus with respect to some variation in the DNA sequence of the fragment. During FA the comparative mobility of DNA fragments is examined as they move under electrophoresis through a gel matrix. There are different applications to FA as seen below.

STR Fragment Analysis

Fragment analysis can be used to determine the genotype of individuals using biomarkers such as microsatellites, also known as Short Tandem Repeats (STR). A microsatellite has a defined repeat unit length. STRs can be a 1-bp homopolymer repeat (...TTTTTTT...), a dinucleotide repeat (....AGAGAGAGAGAGAG.....), trinucleotide repeat (...AGTAGTAGTAGTAGT...) etc. The size of the microsatellite at any given locus may vary from individual to individual due to error during DNA replication caused by the DNA polymerase. These errors or replication slippages generate repeat units that are either longer or shorter than the original unit. Since STRs are in non-coding regions of the genome, changes in their repeat units can accumulate over generations without deleterious effect to the host.

The importance of microsatellites for biological analysis is well established. They are highly polymorphic markers that are dispersed throughout the genomes of many animal and plant species. These abundant markers are widely used in fragment analysis labs for genetic mapping (linkage analysis and association), identity testing, loss of heterozygosity (LOH) and the analysis of genetic diversity in population studies. Due to their high power of discrimination and ease of analysis via polymerase chain reaction (PCR), microsatellites have also become a gold standard in monitoring samples that may contain a mixed cell population. Examples of these types of samples include fetal cells collected for prenatal testing, experimental sample mix-up, forensic crime scene investigations, and monitoring of residual host cells/transplant engraftment for malignant or non-malignant disorders.

Quantitative Fragment Analysis

FA can be used as a quantitative tool to determine the copy number or haplotype of a specific region of the chromosome. In this application a series of discrete DNA fragments are amplified by PCR in multiplex (at the same time, in the same tube). The assay usually includes a single copy gene marker as an internal control. By comparing the signal of the gene-specific PCR products to that of the control one can determine the copy number of the gene of interest. Quantitative PCR fragment analysis is a method well suited to accurately determine the copy number of a transgene. Nemours researchers currently use quantitative FA as a diagnostic tool to identify duplication events in disease causing genes, as well as for the identification of carrier individuals.

Detection of DNA Fragments

The STR and quantitative fragment analysis applications that are currently available through the core laboratory use fluorescent dye technology. During the PCR step one of the primers for each of the loci amplified is labeled with a fluorescent dye. Currently, two genetic analyzers are available for FA: The ABI-PRISM 310 single capillary genetic analyzer and the previously described ABI-PRISM 3130xl 16-capillary array genetic analyzer.

The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of fragment analysis applications. Investigators can perform genotyping for linkage analysis, SNP detection, mutation detection, quantitative analysis and many other applications. Dye-labeled PCR products are electrophoresed through a micro-thin capillary. Products are excited by a laser and detected with a CCD camera.

The ABI 310 supports the use of 4-dye chemistry. It can detect fragments labeled with 3 fluorescently labeled primers plus a size marker ladder labeled with a fourth dye. Because of the distinct spectral signature of the 4 dyes that are included in each set, many PCR products can be run at the same time as exemplified below.

This chromatogram is the display obtained after STR-PCR of an individual's DNA amplified using 10 different primer pairs. The internal size standard labeled with Rox dye is detected in red. The microsatellite-specific DNA fragments are labeled with FAM, JOE and NED dyes, which are detected as blue, green and black respectively.

The ABI PRISM 3130xl genetic analyzer affords higher throughput for FA than the ABI PRISM 310. In addition to the 16-capillary array format of this instrument, it has 5-dye detection capabilities. It can detect fragments labeled with 4 fluorescently labeled primers (FAM, VIC, PET and NED dyes), plus one size marker ladder labeled with the orange dye LIZ.

Nemours Biomedical Research, a division of The Nemours Foundation, operates and in collaboration with the University of Delaware. All content is © Nemours Biomedical Research and The Nemours Foundation unless otherwise noted.