The Nobel Committees have recently announced the much-anticipated Nobel Prize winners of 2022, honouring the contributions that, as per Alfred Nobel’s will of 1895, “have conferred the greatest benefit to humankind”. In this series of articles, we present selected patents of these winners, that, at least to some extent, result from or lead to their celebrated works. For those with an interest in the most commemorated scientific and technological achievements in 2022 and intellectual property, this series makes for interesting reading.
On 3 October 2022, the Nobel Assembly at Karolinska Institutet announced that the 2022 Nobel Prize in Physiology or Medicine was rewarded to Svante Pääbo “for his discoveries concerning the genomes of extinct hominins and human evolution”.
The Committee commended that:
Through his pioneering research, Svante Pääbo accomplished something seemingly impossible: sequencing the genome of the Neanderthal, an extinct relative of present-day humans. He also made the sensational discovery of a previously unknown hominin, Denisova. Importantly, Pääbo also found that gene transfer had occurred from these now extinct hominins to Homo sapiens following the migration out of Africa around 70,000 years ago. This ancient flow of genes to present-day humans has physiological relevance today, for example affecting how our immune system reacts to infections.
Specifically, Pääbo employed ground-breaking techniques that enabled sequencing of ancient human genomes from ancient bone remains containing tiny amount of DNA, which is usually contaminated with DNA from other sources e.g. bacteria and contemporary humans.
Patents
We present herein two patent families with Pääbo listed as one of the inventors. These two families relate to improved, rapid and reliable DNA sequencing methods with a reduction of the initial amount of nucleic acid molecules required, for use in sequence determination in medical diagnostics, forensics and population genetics.
Family 1 – Method for the direct, exponential amplification and sequencing of DNA molecules and its application
The Family 1 patents claim priority from DE19653439 and have a priority date of 20 December 1996. EP0849364, for example, has two independent claims:
1. Method for the direct sequencing of a DNA molecule from a complex mixture of nucleic acids in which truncated as well as DNA molecules of full length are simultaneously synthesised between two positions on the said DNA molecule in a thermocyclic reaction which initially comprises a DNA molecule, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide whereas the ratio of dNTPs: ddNTPs or the other terminating nucleotide respectively is between 100:1 and 1000:1 and, wherein the thermostable DNA polymerase has a reduced discrimination against the four ddNTPs compared to wild-type Taq polymerase.
18. Initial reaction mixture for the direct sequencing of a nucleic acid molecule from a complex mixture of nucleic acids containing a reaction buffer, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide whereas the ratio of dNTPs:ddNTPs or the other terminating nucleotide, respectively is between 100:1 and 1000:1, a nucleic acid molecule, a first primer, a second primer and a thermostable polymerase which has a reduced discrimination against ddNTPs compared to wild-type Taq DNA polymerase.
The family 1 patents disclose a method for sequencing nucleic acids which simultaneously potentiates the exponential amplification of molecules of full length and of molecules of truncated length in the reaction. Direct sequencing means that the nucleic acid fragment to be sequenced is simultaneously amplified and sequenced without interrupting the reaction and without prior amplification of the nucleic acid fragment to be sequenced. Accordingly, the reaction can be carried out more rapidly and with fewer manipulations, which leads to a reduction of the required amount of starting nucleic acid. It was surprisingly found that the use of a DNA polymerase which, in comparison to wild-type Taq DNA polymerase, has a reduced discrimination against the four ddNTPs, enables the simultaneous and exponential synthesis of truncated as well as of full fragments from the start of the cycling reaction. The DNA polymerase may be a Taq DNA polymerase with a Tabor-Richardson mutation which also has no 5′-3′ exonuclease activity such as Taq DNA polymerase (-exo5′-3′) (F667Y).
Family 2 – Method for the uncoupled, direct, exponential amplification and sequencing of DNA molecules with the addition of a second thermostable DNA polymerase and its application
The Family 2 patents claim priority from DE19653494 and have a priority date of 20 December 1996. EP0854196, for example, has two independent claims:
1. Method for sequencing a nucleic acid molecule in a thermocycling reaction which initially comprises a nucleic acid molecule, a first primer, a second primer, a reaction buffer, deoxynucleotides or derivatives thereof and at least one dideoxynucleotide or another terminating nucleotide whereas the ratio of dNTPs : ddNTPs or the other terminating nucleotide, respectively is between 100:1 and 1000:1 and wherein the thermocycling reaction contains at least two thermostable DNA polymerases with different enzyme activities for incorporating dideoxynucleotides.
30. Initial reaction mixture for sequencing a nucleic acid molecule containing a nucleic acid molecule, a first primer, a second primer, a reaction buffer, deoxynucleotides or derivatives thereof and at least one dideoxynucleotide or another terminating nucleotide whereas the ratio of dNTPs : ddNTPs or the other terminating nucleotide, respectively is between 100:1 and 1000:1 and wherein the thermocycling reaction contains at least two thermostable DNA polymerases with different enzyme activities for incorporating dideoxynucleotides.
The family 2 patents also disclose a method for sequencing nucleic acids which simultaneously potentiates the exponential amplification of molecules of full length and of molecules of truncated length in the reaction which, like patent family 1, leads to a reduction of the required amount of starting nucleic acid molecules. Additionally, the method allows an increase in the distance between the positions of the two primers on the nucleic acid molecule to be sequenced, is relatively independent of the distance between the said primers and in general does not require an optimization of the reaction conditions for each DNA fragment to be sequenced. It was surprisingly found that direct, exponential amplification and sequencing can be carried out by adding two different types of DNA polymerases to the initial cycle sequencing reaction: a first thermostable DNA polymerase such as a Taq polymerase and a second thermostable DNA polymerase with a reduced ability to incorporate dideoxynucleotides compared to the said first thermostable DNA polymerase such as a DNA Taq polymerase of the type described in patent family 1. The first DNA polymerase produces truncated products which accumulate exponentially during the cycles and contribute to the sequence ladder that is generated, while the second DNA polymerase produces products of full length which accumulate and serve in subsequent cycles as a template for the production of further DNA strands of full length as well as templates for extensions which contribute to the sequencing reaction. The combination of the different properties of the two polymerases, i e. the ability of the first DNA polymerase to efficiently incorporate dideoxynucleotides and the ability of the second DNA polymerase to efficiently incorporate deoxynucleotides, leads to a considerably increased efficiency of the uncoupled, direct, exponential amplification and sequencing reaction.
Svante Pääbo’s patents and works are well worth a more detailed review for interested parties.