Looking at molecular DNA. Credit: snre/Flickr, CC BY 2.0
Looking at molecular DNA. Credit: snre/Flickr, CC BY 2.0

Featured image credit: snre/Flickr, CC BY 2.0.

Ahead of Eid al Adha celebrations on September 13, the police in Haryana’s Mewat district were tasked with sniffing through morsels of meat biryani sold by vendors to check for the presence of cow beef. Haryana has some of India’s strictest laws on the production and consumption of cow-meat. The state also receives the largest number of complaints against these acts after Uttar Pradesh, according to the National Crime Records Bureau. However, the human senses are easily waylaid, especially when the political climate is charged, allowing room for the sort of arbitrariness that had goons baying for the blood of Mohammad Akhlaq in Dadri in September 2015.

The way to check if a piece of meat is from a cow is to ascertain if it contains cow DNA. The chemical test used for this is called a polymerase chain reaction (PCR), which rapidly creates multiples copies of whatever sample DNA is available and then analyses them according to preprogrammed rules. However, the PCR method isn’t very effective when the DNA might be damaged – such as when the meat is cooked at high temperatures for a long time.

The DNA molecule in most living creatures on Earth consists of a sequence of smaller molecules called nucleotides. The sequence of nucleotides in their entirety is unique to each individual creature as long as its cells contain DNA. A segment of these nucleotides also indicate what species the creature belongs to. It is this segment that a molecular biologist, usually someone at the postgraduate level or higher, will mount a hunt for using the physical and chemical tools at her disposal. The segment’s nucleotides and their ordering will give away the DNA’s identity.

The Veterinary and Animal Sciences University in Hisar, Haryana, is one centre where these tests are conducted. NDTV reported on September 10 that the university had been authorised to do so only two days before it received its first test sample. The vice-chancellor subsequently clarified that two other centres in the state were being set up to conduct these tests – but until they were ready, the university lab would be it.

What would need to be set up? Essentially: an instrument called a thermal cycler to perform the PCR and someone qualified to conduct the PCR, usually at the postgraduate level or higher. The following is how PCR works.

Once some double-strands have been extracted from cells in the meat, they are heated to about 96 ºC for around 25 seconds to denature them. This breaks the bonds holding the two strands together, yielding single strands. Then, two molecules, a primer and a probe, are made to latch onto each DNA single-strand. Primers are small strands of DNA, typically a dozen nucleotides long, that bind complementarily to the single-strand – i.e., the nucleotides adenine on one strand with thymine on the other, and cytosine on one with guanine on the other. Probes are also complementary strands of nucleotides, but its nucleotides are chosen such that the probe binds to sequences that identify the DNA as being from cows. They also contain some fluorescent material.

To enable this latching, the reaction temperature is held at 50-65 ºC for about 30 seconds.

Next, an enzyme called a DNA polymerase is introduced into the reaction solution. The polymerase elongates the primer – by weaving additionally supplied nucleotides along the single-strand to make a double-strand all over again. When the polymerase reaches the probe, it physically disintegrates the probe and releases the fluorescent material. The resulting glow in the solution signals to the researcher that a nucleotide sequence indicative of cow is present in the DNA.

If the Taq polymerase, extracted from microbes living around hot hydrothermal vents on the ocean floor, is used, the reaction temperature is maintained at 72 ºC. In this scenario, the polymerase weaves in about 1,000 nucleotides per minute.

A molecular biologist repeats these three tasks – denaturing the strands, latching the primer and probe on and elongating the primer using polymerase – in repeated cycles to make multiple copies of DNA. At the end of the first cycle, there is one double-strand DNA. At the end of the second, there are two. At the end of the third, there will be eight. So each cycle produces 2n DNA double-strands. When 20 cycles are performed, the biologist will possess over a million DNA double-strands. After 40 cycles, there will be almost 1.1 trillion. Depending on the number of cycles, PCR could take between two and six hours.

These many DNA molecules are needed to amplify their presence, and expose their nucleotides for the finding. The heating cycles are performed in the thermal cycler. This instrument can be modified to track the rate of increase of fluorescence in the solutions, and check if that’s in line with the rate at which new DNA double-strands are made. If the two readings line up, the molecular biologist will have her answer: that the DNA identifies meat from a cow.

The test gets trickier when the meat is cooked. The heat during preparation could damage the DNA in the meat’s cells, denaturing it to a point beyond which PCR can work with. One biologist The Wire spoke to said that if the “meat is nicely overcooked at high temperature, you cannot PCR anything”. A study published in the journal Meat Science in 2006 attests to this: “… with the exception of pan frying for 80 min, beef was determined in all meat samples including the broth and sauce of the roasted meat” using PCR.

At the same time, in March 2016, a study published in Veterinary World claimed that PCR could check for the origins of cooked and raw meat both, and also ascertain the presence of a small amount of beef (up to 1%) present in a larger amount of a different meat. The broader consensus among biologists seems to be that the more raw the meat, the easier it would be to test. The meat starts to become untestable when cooked at high temperatures.

A PCR test costs anywhere between Rs 2,000 and Rs 7,000.

The Wire
September 15, 2016

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