Technologies of genome research
Selected technologies of genome research
Genome researchers are not only working with single genes, but with entire genomes. They are not working with single patients but with patient cohorts, not with single diseases but with disease overlapping mechanisms. Thus, molecular techniques in ordinary laboratory scope are not sufficient for genome research any more.Therefore high throughput approaches were developed, that can generate shortly huge amounts of data. These data can then be translated in evaluable information by the bioinformatics, which role is getting more and more important.
Two of the main methods in genome research are presented in this chapter.
DNA sequencing
DNA-sequencing revolutionized the life sciences and initiated the era of genome research. 1977 about 35 years after the decoding of the DNA structure, two technologies were developed in parallel, that elucidated the sequence of the bases in the DNA.
Fred Sanger developed a method that uses new generated DNA by enzymes, which was subsequently sequenced. The method of Maxam and Gilbert however used a chemical degradation of the DNA. Both, Sanger and Gilbert were 1980 awarded with the Nobel prize of chemistry for their development of sequencing techniques. Especially because of the quality of the sequences, the better reading capacity and because it was easier to automate the process, the sequencing method of Sanger has been established.
The “Next generation sequencing” like the pyrosequencing, a method developed in 1996, provide the possibilities of high-throughput sequencing and are nowadays indispensable in genome research.
For the sequencing according to Allan Maxam and Walter Gilbert, DNA is marked at one end. In four independent approaches one base is modified and removed respectively. Subsequently, the DNA strand is cleaved at the breach. In every approach, fragments in different sizes are generated whose endings always stop at a specific base. Using gel electrophoresis, the fragments are separated by size and detected, whereby differences of one base can be distinguished. The comparison of the four approaches reveals the sequence of the DNA.
In 1975 Frederic Sanger and Alan Coulson developed the 'dideoxy' method of sequencing. In this process, the DNA doublehelix is melted into single strands. Then, using the enzyme DNA polymerase one of the complementary strands is prolonged based on a short known sequence (primer). In four independent approaches each containing all four nucleotides (NTPs), modified nucleotides are added. The incorporation of the modified nucleotides (ddNTPs) lacking the 2' hydroxyl groups required for the addition of the next base causes a random chain termination. So, fragments in different sizes are generated whose endings in each approach always stop at a specific base. By labelling either the primer or the ddNTPs, the the fragments can be separated by gel electrophoreses and detected. The comparison of the four approaches reveals the sequence of the sequenced DNA strand. The corresponding complementary sequence is the sequence of the used single strand DNA template.
Pyrosequencing (one of the „next-generation Sequencing“-technologies) like the Sanger method is using a DNA-polymerase to synthesize a DNA strand. However in this process, the DNA polymerase can be monitored “in action”. Using an elaborated enzyme system, the successful insertion of a single nucleotide is transferred in a light signal and detected. The single strand DNA, that is to be sequenced, serves as template. Based on a short known sequence (primer) the strand is prolonged nucleotide by nucleotide through their controlled adding. If a matching (complementary) NTP is added, a signal is sent, if the NTP does not fit, there is no light signal. In this manner, the DNA sequence can be determined.
Using the„next-generation sequencing“-technologies an entire human genome can be sequenced in 8 weeks for 100.000 dollars – a task that needed in the Human Genome Project (HGP) 13 years, thousands of employees and 300 million dollars.
Fred Sanger developed a method that uses new generated DNA by enzymes, which was subsequently sequenced. The method of Maxam and Gilbert however used a chemical degradation of the DNA. Both, Sanger and Gilbert were 1980 awarded with the Nobel prize of chemistry for their development of sequencing techniques. Especially because of the quality of the sequences, the better reading capacity and because it was easier to automate the process, the sequencing method of Sanger has been established.
The “Next generation sequencing” like the pyrosequencing, a method developed in 1996, provide the possibilities of high-throughput sequencing and are nowadays indispensable in genome research.
For the sequencing according to Allan Maxam and Walter Gilbert, DNA is marked at one end. In four independent approaches one base is modified and removed respectively. Subsequently, the DNA strand is cleaved at the breach. In every approach, fragments in different sizes are generated whose endings always stop at a specific base. Using gel electrophoresis, the fragments are separated by size and detected, whereby differences of one base can be distinguished. The comparison of the four approaches reveals the sequence of the DNA.
In 1975 Frederic Sanger and Alan Coulson developed the 'dideoxy' method of sequencing. In this process, the DNA doublehelix is melted into single strands. Then, using the enzyme DNA polymerase one of the complementary strands is prolonged based on a short known sequence (primer). In four independent approaches each containing all four nucleotides (NTPs), modified nucleotides are added. The incorporation of the modified nucleotides (ddNTPs) lacking the 2' hydroxyl groups required for the addition of the next base causes a random chain termination. So, fragments in different sizes are generated whose endings in each approach always stop at a specific base. By labelling either the primer or the ddNTPs, the the fragments can be separated by gel electrophoreses and detected. The comparison of the four approaches reveals the sequence of the sequenced DNA strand. The corresponding complementary sequence is the sequence of the used single strand DNA template.
Pyrosequencing (one of the „next-generation Sequencing“-technologies) like the Sanger method is using a DNA-polymerase to synthesize a DNA strand. However in this process, the DNA polymerase can be monitored “in action”. Using an elaborated enzyme system, the successful insertion of a single nucleotide is transferred in a light signal and detected. The single strand DNA, that is to be sequenced, serves as template. Based on a short known sequence (primer) the strand is prolonged nucleotide by nucleotide through their controlled adding. If a matching (complementary) NTP is added, a signal is sent, if the NTP does not fit, there is no light signal. In this manner, the DNA sequence can be determined.
Using the„next-generation sequencing“-technologies an entire human genome can be sequenced in 8 weeks for 100.000 dollars – a task that needed in the Human Genome Project (HGP) 13 years, thousands of employees and 300 million dollars.
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