It's in our DNA Part 2 - PCR and markers generate information for fingerprinting and QC

It's in our DNA Part 2 - PCR and markers generate information for fingerprinting and QC
It's in our DNA Part 2 - PCR and markers generate information for fingerprinting and QC

In our previous issue, we took a look at the function and structure of DNA and how it is able to code for the 22 000 different proteins in the human body using just four basic building blocks: adenine, cytosine, thymine and guanine.

This genetic blueprint present in the nucleus of each cell is responsible for the functioning, growth and development of the organism, including all metabolic processes and responses to the external environment (like temperature, light, water and diseases).

Each organism has a unique DNA sequence, arranged into chromosomes, with the entire sequence called the genome. Because this sequence is unique, individuals of the same species can be identified using DNA markers. Much like separating a group of people based on eye colour, hair colour, presence or absence of freckles, height and so on, DNA markers can distinguish between individuals at the DNA level. Why would we want to do this? It is an incredibly powerful tool in research and breeding – the ability to characterize each of our breeding sources and lines with a DNA fingerprint enables us to track each one through the research and development process until they become used in commercial varieties. The same DNA markers can be used for quality control of seed to ensure it is the correct variety and it is a pure source.

How do DNA markers work?

A technique called polymerase chain reaction (PCR) was developed in the mid-1980s that altered the landscape of biology forever. American biochemist Kary Mullis, who won the 1993 Noble prize for developing PCR, used a naturally occurring enzyme present in all organisms – DNA polymerase – to amplify a short stretch of DNA between two DNA primers. The usual role of this enzyme is to copy and replicate DNA from a template – every time one of the cells in your body divides, the DNA in the nucleus needs to be copied so that each new cell can have a full set of the DNA code. DNA polymerase reads the existing DNA and uses it as a template to assemble an exact copy, base by base. When your chromosomes are replicated prior to cell division, multiple DNA polymerase enzymes are simultaneously busy at different locations (each going at 50 bases per second), so that the 150-million bases on one chromosome takes only about one hour to copy.

Mullis used this inherent feature to repeatedly amplify a small region between two defined primers, over and over again (hence chain reaction) using alternating temperatures for different parts of the process (denaturation of double strands, primer annealing, amplification). The two primers that flank the region being amplified are designed based on the unique sequence being targeted, and function like very specific address coordinates (a molecular version of a geotag). Once the short DNA section is amplified, it can be analysed (using a gel, fluorescent detector, or sequencer). Differences within this amplified fragment are used to distinguish individuals. When multiple different markers from different chromosomes are combined, a unique fingerprint can be generated.

There are different types of markers, each generating various levels of information. Some markers give information on the presence or absence of a DNA sequence (binary information), others can distinguish multiple alleles, and are therefore more informative. For a fingerprint to be useful, many markers across all the chromosomes are needed. The densest and most accurate fingerprint is a genome sequence, recording every single base present. A DNA fingerprint for many individuals can be generated simultaneously because the cyclical process of PCR can be automated by machines.

Analysing DNA fingerprints can help breeders understand the relatedness of their breeding populations and allow them to design new combinations for crossing. In many cases, high performing F1 hybrids result from the combination of genetically diverse parents (an effect called hybrid vigour, or heterosis). These benefits extend into the quality control arena so that newly delivered seeds can be analysed to ensure the variety is correct, and the seed is genetically uniform. For example, the seed of most tomato varieties all look the same, and a seed mixture would not be picked up by visually inspecting the seed. However, a DNA fingerprint will be able to distinguish seeds of the correct variety from an off-type variety.

Starke Ayres has a state-of-the-art DNA lab capable of running more than 100 000 PCR reactions each day for analysis. DNA from seed or leaf tissue is extracted using an extraction robot, and these samples are combined with the DNA polymerase and primers for each different marker. The reactions are transferred to a PCR machine that cycles through different temperatures, allowing the enzyme to amplify each targeted region. Once each completed reaction is scanned, our biotechnologists analyse the data and generate reports used by the breeder or QC lab. Our commitment to delivering the highest quality seeds to our customers means we go to great lengths to ensure the genetic purity of each seed lot we produce.

Look out for the third and final instalment, “It’s in our DNA part 3 – Marker-assisted selection: how it works and why we use it”, in the next edition of Seed News.