Polymerase Chain Reaction

Polymerase Chain Reaction DNA amplification and much more

Table of Contents • An introduction to PCR • The PCR process • What is PCR? • Typical components of a PCR • PCR animation • Typical PCR conditions • PCR optimization • Magnesium concentration • Primer annealing temperature • PCR primer design • DNA quality and quantity

Table of Contents • Applications of PCR • Reverse transcription PCR • DNA or RNA labeling • DNA or RNA cloning • DNA and RNA detection • DNA and RNA quantitation • Genotyping and DNA-based identification

An Introduction to PCR • To fully understand cellular processes, scientists often examine events at the molecular level. • Scientific studies often involve: • analysis of DNA, RNA and protein molecules • use of labeled DNA as a molecular tool to visualize these biomolecules • Scientists need a quick and easy way to produce DNA in sufficient quantities for their studies and generate labeled DNA molecules to visualize and study specific molecules within cells.

What is PCR? • The polymerase chain reaction (PCR) is a relatively simple technique developed in 1985 to amplify sequence-specific DNA fragments in vitro. • PCR is one of the most useful techniques in laboratories today due to its speed and sensitivity. • Traditional techniques to amplify DNA require days or week. PCR can be performed in as little as 1 hour. • Many biochemical analyses require the input of significant amounts of biological material; PCR requires as little as one DNA molecule. • These features make PCR extremely useful in basic research and commercial applications, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics.

PCR Amplifies a Specific DNA Sequence • PCR can be used to target a specific DNA subsequence in a much larger DNA sequence (e.g., a single 1000bp gene from the human genome, which is 3 × 109bp). • PCR allows exponential amplification of a DNA sequence. • Each PCR cycle theoretically doubles the amount of DNA. • During PCR, an existing DNA molecule is used as a template to synthesize a new DNA strand. • Through repeated rounds of DNA synthesis, large quantities of DNA are produced.

The PCR Process—Reaction Components Typical components of a PCR include: • DNA: the template used to synthesize new DNA strands. • DNA polymerase: an enzyme that synthesizes new DNA strands. • Two PCR primers: short DNA molecules (oligonucleotides) that define the DNA sequence to be amplified. • Deoxynucleotide triphosphates (dNTPs): the building blocks for the newly synthesized DNA strands. • Reaction buffer: a chemical solution that provides the optimal environmental conditions. • Magnesium: a necessary cofactor for DNA polymerase activity.

The PCR Process—PCR Primers PCR primers • Primers define the DNA sequence to be amplified—they give the PCR specificity. • Primers bind (anneal) to the DNA template and act as starting points for the DNA polymerase, since DNA polymerases can only extend existing DNA molecules and cannot initiate DNA synthesis without a primer. • The distance between the two primers determines the length of the newly synthesized DNA molecules.

How does PCR Amplify DNA? • One PCR cycle consists of a DNA denaturation step, a primer annealing step and a primer extension step. DNA Denaturation: Expose the DNA template to high temperatures to separate the two DNA strands and allow access by DNA polymerase and PCR primers. Primer Annealing: Lower the temperature to allow primers to anneal to their complementary sequence. Primer Extension: Adjust the temperature for optimal thermostable DNA polymerase activity to extend primers. • PCR uses a thermostable DNA polymerase so that the DNA polymerase is not heat-inactivated during the DNA denaturation step. Taq DNA polymerase is the most commonly used DNA polymerase for PCR.

PCR Animation View the PCR animation for a dynamic PCR demonstration.

Mechanism of DNA Synthesis • DNA polymerase extends the primer by sequentially adding a single dNTP (dATP, dGTP, dCTP or dTTP) that is complementary to the existing DNA strand • The sequence of the newly synthesized strand is complementary to that of the template strand. • The dNTP is added to the 3´ end of the growing DNA strand, so DNA synthesis occurs in the 5´ to 3´ direction.

Instrumentation • Thermal cyclers have a heat-conducting block to modulate reaction temperature. • Thermal cyclers are programmed to maintain the appropriate temperature for the required length of time for each step of the PCR cycle. • Reaction tubes are placed inside the thermal cycler, which heats and cools the heat block to achieve the necessary temperature.

Thermal Cycling Programs A typical thermal cycler program is: Initial DNA denaturation at 95°C for 2 minutes 20–35 PCR cycles: Denaturation at 95°C for 30 seconds to 1 minute Annealing at 42–65°C for 1 minute Extension at 68–74°C for 1–2 minutes Final extension at 68–74°C for 5–10 minutes Soak at 4°C

PCR Optimization Many PCR parameters might need to be optimized to increase yield, sensitivity of detection or amplification specificity. These parameters include: • Magnesium concentration • Primer annealing temperature • PCR primer design • DNA quality • DNA quantity

Magnesium Concentration • Magnesium concentration is often one of the most important factors to optimize when performing PCR. • The optimal Mg2+ concentration will depend upon the primers, template, DNA polymerase, dNTP concentration and other factors. • Some reactions amplify equally well at a number of Mg2+ concentrations, but some reactions only amplify well at a very specific Mg2+ concentration. • When using a set of PCR primers for the first time, titrate magnesium in 0.5 or 1.0mM increments to empirically determine the optimal Mg2+ concentration.

Primer Annealing Temperature • PCR primers must anneal to the DNA template at the chosen annealing temperature. • The optimal annealing temperature depends on the length and nucleotide composition of the PCR primers • The optimal annealing temperature is often within 5°C of the melting temperature (Tm) of the PCR primer The melting temperature is defined as the temperature at which 50% of complementary DNA molecules will be annealed (i.e., double-stranded). • When performing multiplex PCR, where multiple DNA targets are amplified in a single PCR, all sets of PCR primers must have similar annealing temperatures.

PCR Primer Design Many PCR failures can be avoided by designing good primers. • Ideally all primers used in a PCR will have similar melting temperatures and GC content. Typically primers with melting temperatures in the range of 45–70°C are chosen. GC content should be near 50%. • Primers should have little intramolecular and intermolecular secondary structure, which can interfere with primer annealing to the template. • Primers with intramolecular complementarity can form secondary structure within the same primer molecule. • Intermolecular complementarity allows a primer molecule to anneal to another primer molecule rather than the template. • Software packages exist to design primers.

DNA Quality • DNA should be intact and free of contaminants that inhibit amplification. • Contaminants can be purified from the original DNA source. • Heme from blood, humic acid from soil and melanin from hair • Contaminants can be introduced during the purification process. • Phenol, ethanol, sodium dodecyl sulfate (SDS) and other detergents, and salts.

DNA Quantity DNA quantity • More template is not necessarily better. • Too much template can cause nonspecific amplification. • Too little template will result in little or no PCR product. • The optimal amount of template will depend on the size of the DNA molecule.

DNA DNA DNA DNA RNA cDNA PCR DNA DNA Reverse transcription DNA DNA DNA Reverse Transcription PCR • RNA can be amplified by including a simple reverse transcription (RT) step prior to PCR. This process is known as RT-PCR. • Reverse transcriptases are RNA-dependent DNA polymerases, which use an RNA template to make a DNA copy (cDNA). This cDNA can be amplified using PCR.

RT-PCR Components Typical components of an RT-PCR include: • Reverse transcriptase: the enzyme that synthesizes the cDNA copy of the RNA target. • Reverse transcription primer: a single short DNA molecule that acts as starting points for the reverse transcriptase, since reverse transcriptases cannot initiate DNA synthesis without a primer. • Deoxynucleotide triphosphates (dNTPs): the building blocks for the newly synthesized cDNA. • Reaction buffer: a chemical solution that provides the optimal environmental conditions. • Magnesium: a necessary cofactor for reverse transcriptase activity. • All of the necessary PCR components for the PCR portion of RT-PCR.

Applications of PCR PCR and RT-PCR have hundreds of applications. In addition to targeting and amplifying a specific DNA or RNA sequence, some common uses include: • Labeling DNA or RNA molecules with tags, such as fluorophores or radioactive labels, for use as tools in other experiments. • Cloning a DNA or RNA sequence • Detecting DNA and RNA • Quantifying DNA and RNA • Genotyping and DNA-based identification

Labeling DNA • Labeling DNA with tags for use as tools (probes) to visualize complementary DNA or RNA molecules. • Radioactive labels. • Radioactively labeled probes will darken an X-ray film. • Fluorescent labels (nonradioactive) • Fluors will absorb light energy of a specific wavelength (the excitation wavelength) and emit light at a different wavelength (emission wavelength). • The emitted light is detected by specialized instruments such as fluorometers.

Identifying a DNA Sequence • To study a specific DNA sequence, that DNA sequence must be targeted and isolated (cloned) so that other surrounding DNA sequences do not interfere with the studies. • PCR is faster and less labor-intensive than traditional cloning techniques. • Traditional cloning techniques require weeks or months. PCR requires hours. • PCR primers can be designed to amplify the exact sequence of interest. • Traditional cloning techniques do not always yield the exact DNA fragment of interest.

DNA and RNA Detection • PCR can detect foreign DNA sequences in a biological sample. • Example: Hospitals often use PCR to detect bacteria and viruses and help diagnose illnesses. • PCR can detect specific DNA sequences to characterize an organism. • Example: The multidrug resistance (MDR) gene confers resistance to antibiotics that are commonly used to treat bacterial infections. PCR using primers specific for the MDR gene will identify strains of bacteria that express MDR and are resistant to common antibiotics. • RT-PCR can detect specific RNA sequences within a sample. • Example: Retroviruses have an RNA genome. Retroviral RNA can be detected by RT-PCR to diagnose retroviral infections.

DNA and RNA Quantitation • Quantitative PCR can be used to determine the copy number of a DNA sequence such as a gene within a genome or the number of organisms present in a sample (e.g., determining viral load). • Quantitative RT-PCR is often used to quantitate the level of messenger RNA (mRNA) produced in a cell. • As gene expression within a cell is activated or repressed, the level of corresponding mRNA increases or decreases, respectively. • Quantitating mRNA levels by RT-PCR can tell us which genes are being up- or downregulated under certain conditions, providing insight into gene function.

Quantitative PCR • Avoids problems associated with the plateau effect, which reduces amplification efficiency and limits the amount of PCR product generated due to depletion of reactants, inactivation of DNA polymerase and accumulation of reaction products. • The result of the plateau effect is that the amount of PCR product generated is no longer proportional to the amount of DNA starting material. • The plateau effect becomes more pronounced at higher cycle numbers. • Often performed in real time to monitor the accumulation of PCR product at each cycle. • Real-time PCR allows scientists to quantify DNA before the plateau effect begins to limit PCR product synthesis.

Genotyping and DNA-Based Identification • Cellular (genomic) DNA contains regions of variable sequences that differ between strains or even individual organisms. • Variable regions are amplified by multiplex PCR, and when the resulting DNA fragments are separated by size, the resulting pattern acts like a unique barcode to identify a strain or individual. • For human identification, these variable regions often include short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs). • STRs and SNPs are useful in DNA-based forensic investigations, missing persons investigations and paternity disputes.

DNA-Based Human Identification The police collect a hair from a crime scene and submit it for STR analysis (sample #1). Five suspicious people were observed near the crime scene shortly after the crime was committed. The police collect DNA from these five people and submit it for STR analysis (samples #2–6). Do any of these five DNA samples match the DNA from the hair collected at the cime scene?

Polymerase Chain Reaction