Realtime Pcr Analysis

There are three distinct phases that define the PCR process: geometric or exponential amplification, linear amplification, and the plateau region (Bloch 1991). These regions can be seen in a plot of fluorescence versus PCR cycle number (Figure 4.5). During exponential amplification, there is a high degree of precision surrounding the production of new PCR products. When the reaction is performing at close to 100% efficiency, then a doubling of amplicons occurs with each cycle (see Table 4.1). A plot of cycle number versus a log scale of the DNA concentration should result in a linear relationship during the exponential phase of PCR amplification.

A linear phase of amplification follows the exponential phase as one or more components fall below a critical concentration and amplification efficiency

Figure 4.5

Real-time PCR output and example standard curve used to determine quantity of input DNA.

slows down to an arithmetic increase rather than the geometric one in the exponential phase. Since components such as dNTPs or primers may be used up at slightly different rates between reactions, the linear phase is not as precise from sample-to-sample and therefore is not as useful for comparison purposes.

The final phase of PCR is the plateau region where accumulation of PCR product slows to a halt as multiple components have reached the end of their effectiveness in the assay. The fluorescent signal observed in the plateau phase levels out. The accumulation of PCR product generally ceases when its concentration reaches approximately 10-7M (Bloch 1991).

The optimal place to measure fluorescence versus cycle number is in the exponential phase of PCR where the relationship between the amount of product and input DNA is more likely to be consistent. Real-time PCR instruments use what is termed the cycle threshold (CT) for calculations. The CT value is the point in terms of PCR amplification cycles when the level of fluorescence exceeds some arbitrary threshold, such as 0.2, that is set by the real-time PCR software to be above the baseline noise observed in the early stages of PCR. The fewer cycles it takes to get to a detectable level of fluorescence (i.e., to cross the threshold set by the software), the greater the initial number of DNA molecules put into the PCR reaction. Thus a plot of the log of DNA concentrations versus the CT value for each sample results in a linear relationship with a negative slope (Figure 4.5).

If e the doubles the target copy number (No) with each cycle (C).


The cleavage of TaqMan probes or binding of SYBR Green intercalating dye to double-stranded DNA molecules results in an increase in fluorescence signal. This rise in fluorescence can be correlated to the initial DNA template amounts when compared with samples of known DNA concentration. For example in Figure 4.5, five samples (a,b,c,d,e) are used to generate a standard curve based on their measured CT values. Provided that there is good sample-to-sample consistency and precision, a sample with an unknown DNA quantity can be compared to this standard curve to calculate its initial DNA template concentration. Several published real-time PCR assays and commercial kits, such as the Quantifiler Human DNA Quantification Kit, are briefly reviewed in Table 3.3.


The sensitivity of PCR necessitates constant vigilance on the part of the laboratory staff to ensure that contamination does not affect DNA typing results. Contamination of PCR reactions is always a concern because the technique is very sensitive to low amounts of DNA. A scientist setting up the PCR reaction can inadvertently add his or her own DNA to the reaction if he or she is not careful. Likewise, the police officer or crime scene technician collecting the evidence can contaminate the sample if proper care is not taken. For this reason, each piece of evidence should be collected with clean tweezers or handled with disposable gloves that are changed frequently.

To aid discovery of laboratory contamination, everyone in a forensic DNA laboratory is typically genotyped in order to have a record of possible contaminating DNA profiles. This is often referred to as a staff elimination database (see Chapter 7). Laboratory personnel should be appropriately gowned during interactions with samples prior to PCR amplification (Rutty et al. 2003). The appropriate covering includes lab coats and gloves as well as facial masks and hairnets to prevent skin cells or hair from falling into the amplification tubes. These precautions are especially critical when working with miniscule amounts of sample or sample that has been degraded (see Chapter 7).

Some tips for avoiding contamination with PCR reactions in a laboratory setting include:

■ Pre- and post-PCR sample processing areas should be physically separated. Usually a separate room or a containment cabinet is used for setting up the PCR amplification reactions.

■ Equipment, such as pipettors, and reagents for setting up PCR should be kept separate from other laboratory supplies, especially those used for analysis of PCR products.

■ Disposable gloves should be worn and changed frequently.

■ Reactions may also be set up in a laminar flow hood, if available.

■ Aerosol-resistant pipette tips should be used and changed on every new sample to prevent cross-contamination during liquid transfers.

■ Reagents should be carefully prepared to avoid the presence of any contaminating DNA or nucleases.

■ Ultraviolet irradiation of laboratory PCR setup space when the area is not in use and cleaning workspaces and instruments with isopropanol and/or 10% bleach solutions help to insure that extraneous DNA molecules are destroyed prior to DNA extraction or PCR setup (Kwok and Higuchi 1989, Prince and Andrus 1992).

PCR product carryover results from amplified DNA contaminating a sample that has not yet been amplified. Because the amplified DNA is many times more concentrated than the unamplified DNA template, it will be preferentially copied during PCR and the unamplified sample will be masked. The inadvertent transfer of even a very small volume of a completed PCR amplification to an unamplified DNA sample can result in the amplification and detection of the 'contaminating' sequence. For this reason, the evidence samples are typically processed through a forensic DNA laboratory prior to the suspect reference samples to avoid any possibility of contaminating the evidence with the suspect's amplified DNA.

Pipette tips should never be reused. Even a tiny droplet of PCR product left in a pipette tip contains as many as a billion copies of the amplifiable sequence. By comparison, a nanogram of human genomic DNA contains only about 300 copies of single-copy DNA markers (see D.N.A. Box 3.3).


We conclude this chapter by reviewing the advantages and disadvantages of PCR amplification for forensic DNA analysis. The advantages of PCR amplification for biological evidence include the following:

■ Very small amounts of DNA template may be used from as little as a single cell.

■ DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification.

■ Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions.

■ Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.

■ Commercial kits are now available for easy PCR reaction setup and amplification.

There are three potential pitfalls that could be considered disadvantages of PCR:

1. The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA (see Chapter 7 discussion on PCR inhibition).

2. Amplification may fail due to sequence changes in the primer-binding region of the genomic DNA template (see Chapter 6 discussion on null alleles).

3. Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols (see Chapter 7 discussion on PCR contamination and Chapter 16 on laboratory validation).


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