Getting data from PCR

Once a particular gene region has been amplified from the requisite number of samples, a genetic identity must be assigned to each individual. The simplest way to do this is from the size of the amplified product, which can be quantified by running out the completed PCR reaction on an agarose or acrylamide gel following the same principle of gel electrophoresis that is used for allozymes. The gel solutions are made up in liquid form, and combs are left in place while the gel is hardening, after which time the combs are removed to leave a solid gel with a number of wells at one end. The DNA samples are loaded into the wells and an electrical field is applied. The DNA molecules are negatively charged and therefore will migrate towards the positive electrode.

The speed at which fragments of DNA migrate through electrophoresis gels depends primarily on their size, with the largest fragments moving most slowly. Once DNA fragments have segregated across a gel, they can be visualized using a dye called ethidium bromide, which binds to DNA molecules and can be seen with the human eye when illuminated with short-wavelength ultraviolet light. The sizes of DNA bands then can be extrapolated from ladders that consist of DNA fragments of known sizes (Figure 1.10). If the amplified products are of variable

Negative electrode —

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6

1 1 1 1 1 1 1 1 1 1 1 --Wells into which samples are loaded

es 400

s er 300

ar m

Positive electrode -)—

Figure 1.10 Representation of an agarose gel after DNA fragments have been stained with ethidium bromide. Lanes 1 and 6 are size markers; note that the smaller fragments migrate through the gel more rapidly, and therefore further, than the larger fragments, which is why fragments of different sizes will separate at a predictable rate. The samples in lanes 2 and 5 have a single band of just over 400 bases long. The sample in lane 3 has two bands that are both close to 200 bases long, and in lane 4 the two bands are close to 100 and 300 bases long sizes then at this stage we may be able to assign individual genetic identities. However, it is often the case that sequences with different compositions will be of the same length, in which case sequencing the PCR products will allow us to identify different genotypes.

DNA Sequencing

The most common method of DNA sequencing, known as dideoxy or sanger sequencing, was invented by Frederick Sanger in the mid-1970s (work that helped him to win a shared Nobel prize in 1980). His protocol was designed to synthesize a strand of DNA using a DNA polymerase plus single nucleotides in a manner analogous to PCR, but with two important differences. First, only a single primer is used as the starting point for synthesis so that sequences are built along the template in only one direction. Second, some of the nucleotides contain the sugar dideoxyribose instead of deoxyribose, the sugar normally found in DNA. Dideoxyribose lacks the 30-hydroxyl group found in deoxyribose, and without this the next nucleotide cannot be added to the growing DNA strand; therefore, whenever a nucleotide with dideoxyribose is incorporated into the reaction, synthesis will be terminated.

Dideoxy sequencing can be done in four separate reactions, each of which include all four nucleotides in their deoxyribose form (dNTPs) and a small amount of one of the nucleotides (G,A,T or C) in its dideoxyribose form (ddNTP). Incorporation of the ddNTPs eventually will occur at every single site along the DNA sequence, resulting in fragment sizes that represent the full spectrum from 1 bp of the target sequence to its maximum length. Different fragment sizes will be generated by each of the four reactions, and in manual sequencing the products of each reaction are run out in separate but adjacent lanes on a gel. Fragments can be visualized in a number of ways, including silver staining or the use of radioactive labels (isotopes of sulfur or phosphorus) that can be developed on x-ray films following a process known as autoradiography. All of the fragment sizes in a given lane indicate positions at which the dideoxyribose bases for that particular reaction were incorporated. For example, if the reaction containing the dideoxy form of dATP contains fragments that are 1, 5, and 10 bp long, then the first, fifth and tenth bases in the sequence must be adenine (A). The fragments from each of the four reactions can be pieced together to recreate the entire sequence (Figure 1.11).

AGGCATCGTA

AGGCATCGT

AGGCATCG

AGGCATC

AGGCAT

AGGCA

AGGC

Figure 1.11 Representation of a sequencing gel. Reactions were loaded into the lanes labeled G, A, T and C, depending on which nucleotide was present in the dideoxyribose form. Because the smallest fragments migrate most rapidly, we can work from the bottom to the top of the gel to generate the cumulative sequences that are shown on the right-hand side

AGGCATCGTA

AGGCATCGT

AGGCATCG

AGGCATC

AGGCAT

AGGCA

AGGC

Figure 1.11 Representation of a sequencing gel. Reactions were loaded into the lanes labeled G, A, T and C, depending on which nucleotide was present in the dideoxyribose form. Because the smallest fragments migrate most rapidly, we can work from the bottom to the top of the gel to generate the cumulative sequences that are shown on the right-hand side

Although manual dideoxy sequencing was the norm for a number of years, it is now being replaced in many universities and research institutions by automated sequencing. Many brands and models of automated sequencers are currently available but the principle remains the same in all. The different fragments that make up a sequence are generated as before, but the nucleotides that contain dideoxyribose are labelled with different-coloured fluorescent dyes. The reactions do not need to be kept separate in the same way as they do with manual sequencing, because different colours represent the size fragments that were terminated by each type of ddNTP. When these reactions are run out on automated sequencers, lasers activate the colour of the fluorescent label of each band (typically black for G, green for A, red for T and blue for C). Each colour is then read by a photocell and stored on a computer file that records the fragments as a series of different-coloured peaks. By substituting the appropriate base for each coloured peak, the entire sequence can be read from a single image.

Real-time PCR

So far we have looked at how PCR can provide valuable genotypic information from the sizes or sequences of amplified products. One thing that conventional PCR cannot do, however, is to supply us with accurate estimates of the amount of DNA that is present in a particular sample. This is because there is no correspondence between the amount of template at the start of the reaction and the amount of DNA that has been amplified by the end of the reaction. In the 1990s, however, a technique known as real-time PCR (RT-PCR, also known as quantitative PCR) was developed, and this does allow researchers to quantify the amount of DNA in a particular sample.

Real-time PCR allows users to monitor a PCR reaction in 'real time', i.e. as it occurs, instead of waiting until all of the cycles in a PCR reaction have finished. The fragments produced in RT-PCR are labelled by either fluorescent probes or DNA binding dyes and can be quantified after each cycle. There is a correlation between the first significant increase in the amount of PCR product and the total amount of the original template. Real-time PCR can quantify DNA or RNA in either an absolute or a relative manner. Absolute quantification determines the number of copies that have been made of a particular template, usually by comparing the amount of DNA generated in each cycle to a standard curve based on a sample of known quantity. Relative quantification allows the user to determine which samples have more or less of a particular gene product.

There are several ways in which quantitative PCR can benefit ecological studies. For one thing, it can provide important insight into the relationship between gene expression and the development of particular phenotypic attributes. Since RNA is transcribed only during gene expression, the amount of RNA in a sample is indicative of the amount of gene expression that is taking place. Researchers can synthesize DNA from an RNA template using the enzyme reverse transcriptase (such 'reverse-engineered' DNA is called complementary DNA, or cDNA). Real-time PCR then can use cDNA as a basis for quantifying gene expression because the amount of cDNA in a sample will be directly proportional to the amount of gene expression that has occurred. Quantitative PCR has been used to identify overexpression of 19 different genes in oysters (Crassostrea virginica and C. gigas) that had been infected with the protozoan pathogen Perkinsus marinus, compared with those that were uninfected (Tanguy, Guo and Ford, 2004), and also to identify variable levels of expression in several key genes that promote salt tolerance in the Euphrates poplar tree Populus euphratica (Gu et al., 2004).

Another application for Real-Time PCR in molecular ecology involves estimation of the numbers (as opposed to simply the identities) of different species within a composite DNA sample. This was done in a study of two species of branching corals, Acropora tenuis and A. valida, living in the Great Barrier Reef. Researchers wished to identify which species of symbiotic algae (zooanthellae) were living within the coral colonies. The identity of these zooanthellae is of interest because they are apparently essential for the maintenance of healthy shallow tropical coral reefs. Bleaching, which is a major threat to coral reefs, occurs when the symbiotic algae living in coral die or lose their pigment because of stresses such as elevated sea temperatures or pollutants, and the resistance of coral reefs to bleaching may be influenced by which species of algae live within coral colonies.

In this particular study the researchers extracted DNA from the coral colonies and used primers specific for different species of the zooanthellae genus Symbio-dinium to identify which zooanthellae were living within different colonies. By using quantitative PCR they were able to determine not only which Symbiodinium species were present but also the extent to which various Symbiodinium species occurred in different coral colonies. These data revealed a relationship between the abundance of Symbiodinium species and the availability of light, which suggests that local adaptation plays a role in the distribution of genetically distinct zooanthellae (Ulstrup and Van Oppen, 2003).

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