Editor's note: We are pleased to present the second of a three-part essay in celebration of 60 years since the discovery of the double helix. You can use the navigation links above right to catch up.

If Watson and Crick were the fathers of molecular biology, many other achievements signalled its coming of age.

Several contributions were crucial for the development of molecular biology, notably the discovery of restriction enzymes (a.k.a. nucleases), thanks to which it is possible to manipulate DNA [16], of DNA sequencing methods [17], and of the polymerase chain reaction (PCR), a protocol to amplify a specific DNA region [18]. All these achievements were awarded the Nobel Prize.

Nucleases are enzymes discovered in the ‘60s which can cut DNA at dedicated sites [16]. These are mostly palindromic (backward-readable) sequences specifically recognized by their symmetry. Was it a car or a cat I saw? is an example of palindrome in English. In the DNA language, an example is GAATTC, if you consider the reverse complement of the DNA sequence – that is, due to pairing of complementary strands, G becomes C, A becomes T, and so on. Since the strands are antiparallel, read it backwards: et voilà! Restriction enzymes made it possible to cut – and then re-ligate (join) them to other pieces of DNA using other enzymes – which made possible to perform genetic engineering and molecular cloning, fundamental techniques in countless molecular biology laboratories around the world.

The discoverers were like copy-and-paste writers, able to take DNA from a book and recombine it into another.

DNA sequencing was introduced in the ‘70s by Frederick Sanger [17] who previously won the Nobel for the development of protein sequencing. Even though the structure of DNA had been known for many years, that didn’t mean it was easy to read its string of nucleotides (the basic DNA unit, composed of a nucleotide bases plus sugar plus phosphate), among other reasons due to its extreme length.

If Watson and Crick were linguists, then Sanger was The Reader. The method he developed was based on the chain-termination method: with a mixture of DNA polymerase (the enzyme responsible for DNA replication), reagents and “fake” nucleotides, it is possible to interrupt the synthesis of a complementary DNA strand (which, when made its reverse complement, read as the string of bases in the DNA strand being sequenced) at every nucleotide. This results in as many DNA strands as the length, in nucleotide bases, of the original one, which when separated by size can be read one after another to deduce the DNA sequence, and in turn, to infer the RNA sequence (and thanks to the genetic code the sequence of amino acids of the resulting protein). If you know some foreign language, read books in the original language – it is more rewarding.

This old-school sequencing method has been ousted by more sophisticated protocols (sequencing-by-synthesis or next-generation sequencing) which are based on detecting the incorporation of nucleotides in a growing complementary strand, which reduced the efforts and costs dramatically [19]. However, Sanger sequencing was still used for the Human Genome Project, declared completed in April 2003, to decode the complete human genome (the total DNA in a cell) [19-20].

PCR (born 1983) is as widespread and essential as the former techniques: with a DNA template, polymerase and a pair of primers (short strings of chemically-synthesized nucleotides which specifically bind at the borders of the soon-to-be-amplified DNA stretch), combined with a thermal cycler to make possible the different reaction steps, it is possible to multiply a desired DNA segment in an exponential way [18].

Its developer Kary Mullis is the Johannes Gutenberg of molecular biology – just a little higher [21]. As he admitted (or staged) in a video interview, he wouldn’t have discovered PCR without the molecule synthesized by the Swiss chemist Albert Hoffmann, the father of LSD [22]. Previous seminal groundwork about amplification of nucleotide stretches by Indian biochemist Har Gobind Khorana [23-24], sharing the Nobel for his work on the genetic code, were neither mentioned in Mullis’ memoir Dancing Naked in the Mind Field nor, as far as I know, anywhere else [21, 25-26].

DNA is the Esperanto of living cells. So, different DNA equals different cells. As simple as that – and as complex as that, too, because every cells in a given organisms has the same DNA, but the cells are different in shape, size and function; also, similarity in DNA sequences of different organisms is much higher of what one might expect by looking at them (the human genome has, for example, 98.8% identity with the chimpanzee’s, while the relationship only drops to 97.5% with that of the mouse).

Concomitant with the advance in our understanding of molecular biology, scientists became increasingly aware that not only are the differences in DNA sequences crucial, but equally crucial – and still mostly obscure even today – is how DNA expression is regulated to give such enormous differences in phenotype (the overall observable traits of an organism). In other words, the more knowledge is gathered on genome biology, the more the “central dogma of molecular biology” is challenged [27]. An early example of that was the discovery of an enzyme in viruses able to invert the flow of information and reverse-transcribe the text from RNA to DNA [28-29]. Another one was the finding of regulatory RNAs in the late ‘90s – that is, RNA is not simply an intermediate in the flux of information between DNA and proteins, but some dedicated RNA molecules can actively influence protein synthesis by a specific mechanism called RNA interference [30]. Guess what: both discoveries got the Nobel.

In both cases the “dogma” was adapted to fit the new findings, but the complex links between genotype (the overall genetic package of an organism) and phenotype are still not fully cracked. Interacting networks governing gene expression are getting so tangled up that the definition of “gene” itself (originally introduced by Mendel and adapted by the “RNA Tie Club” with the simplistic definition of a DNA text coding for a protein) might need revision [27].

Today, what are some of the recent advancements in molecular biology, and why is it that the old grandpa of DNA is still interesting enough to have a chat over a pint about its possible uses? I’ll keep that for the next time.

To be continued...

References:

[16] http://www.annualreviews.org/doi/pdf/10.1146/annurev.bi.38.070169.002343
[17] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/pdf/pnas00043-0271.pdf
[18] http://www.sciencedirect.com/science/article/pii/0076687987550236
[19] http://www.nature.com/jid/journal/v133/n8/pdf/jid2013248a.pdf
[20] http://en.wikipedia.org/wiki/Human_Genome_Project
[21] http://www.amazon.com/Dancing-Naked-Mind-Field-Mullis/dp/0679774009
[22] http://www.youtube.com/watch?v=CC5ApU4YKBU
[23] http://www.sciencedirect.com/science/article/pii/0022283671904694#
[24] http://www.nature.com/milestones/miledna/full/miledna11.html
[25] http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html
[26] http://www.nobelprize.org/mediaplayer/index.php?id=428
[27] http://www.nature.com/nature/journal/v496/n7446/pdf/496419a.pdf
[28] http://www.nature.com/nature/journal/v226/n5252/pdf/2261209a0.pdf
[29] http://www.nature.com/nature/journal/v226/n5252/pdf/2261211a0.pdf
[30] http://www.nature.com/nature/journal/v391/n6669/pdf/391806a0.pdf