How to measure DNA in urine

Standard DNA detection technique

The method of choice for DNA quantification in most laboratories is UV-spectrophotometry. This method is based on the absorption of ultraviolet (UV) light by DNA. Each base (adenine (A), thymine (T), guanine (G) and, cytosine (C)) has an aromatic (ring-like) structure that absorbs UV light.

The absorption maxima of the different nucleotides give an absorption pattern for DNA with a maximum of 260 nm. The amount of absorption can be directly correlated to the amount of DNA in the solution. This method, however, does not give any information on the sequence or level of methylation of the DNA in the sample.

Sequence-specific measurements

Polymerase chain reaction (PCR) is a technique that can give information about the DNA sequence. PCR is a reaction that exponentially multiplies specific parts of a DNA strand (i.e. in every step the number of copies is doubled) by cycling the sample through different temperature steps:

1. Denaturation (95°C): heating the sample to break the hydrogen bonds between the two strands, such that single-stranded DNA is created.
2. Primer annealing (55-60°C): short complementary single-stranded DNA strands (the base pair sequences perfectly complements the single strand sequence, i.e. A to T, C to G and vice versa) of 15-25 bases, called primers, bind highly selective to the DNA sequence of interest. These primers can be designed to bind only to the gene region of interest. This step gives PCR its high specificity for DNA sequence determination.
3. Polymerase elongation (68 °C): the polymerase enzyme binds to the DNA part where the primer is bound since that is the only part of double-strand DNA in the sample. Then it starts to elongate the primer into a new DNA strand, based on the sequence of the single-strand DNA the primer is bound to. By cycling the sample through the different temperatures many times, it will multiply a single DNA copy (which is extremely difficult to measure) into many copies, which can be measured easily. And due to the specificity of the primer sequence that is used, the presence of a specific gene can be investigated.

Methylation-specific PCR

PCR however, does not give information on the methylation of the DNA. Therefore the DNA must be given a specific treatment prior to the PCR: bisulfite conversion.

This reaction targets cytosine and converts it to uracil. Uracil normally does not occur in DNA (it is present in RNA). Methylated cytosines, however, are not susceptible to bisulfite binding and will not be converted. Therefore, if bisulfite conversion and PCR are done successively, PCR primers need to be different for unmethylated (all cytosine converted) and methylated (methylated cytosines remain) regions.

By choosing the right primer sequences, the PCR can tell whether the amplified region has methylation. However, the bisulfite reaction in total takes 4-16 hours.

Next-generation sequence-specific DNA detectors

The next generation of DNA detectors will not need constant cycling of temperatures, in order to amplify the DNA. Advances in microfluidics allow small volumes of DNA samples to be denatured rapidly in microchannels. These single-stranded DNA fragments can then be led over a surface covered with different DNA probing sequences.

The probing sequences are designed to hybridize only with specific sequences of DNA that are of interest and in that way can differentiate between genes. On one detector, multiple types of probes can be immobilized on different spots, allowing one sample to be directly screened for multiple different DNA sequences.

Unfortunately, this, by itself, is not methylation-specific.

Disadvantages of urine

Urine is not a clean sample: it is quite variable inconsistency, depending on the fluid intake of the person. Furthermore, it has a high concentration of salts, organic compounds and proteins.

The presence of these compounds in urine strongly influences the outcome of all measurement techniques.

• First, UV-spectrophotometry is disrupted by the absorption spectra of many organic compounds and proteins present in urine.
• Second, salts, organics and proteins can inhibit PCR.
• Third, the hybridization of DNA to the probing sequences is disrupted by salt (in case the concentration is too high or too low).
• Finally, urine contains enzymes that will break down the DNA, which complicates the detection

All these variables make it very difficult to get a correct measurement of DNA concentration directly in urine.