Definition & background
What is DNA?
The presence of DNA was first discovered by Friedrich Miescher back in 1869 while the structure of DNA molecules as a helical and double stranded was deciphered by Watson and Crick in 1953. In addition, the DNA molecules are treated as the storage of genetic information and acted as the hereditary molecules proved by Avery, Maclead, and McCarty through laboratory demonstration using virus and E. coli (1). The result is consistent to Gregor Mendel’s laws of inheritance that had been proposed in 1865 as gene pair is the inherited traits of organism that led to evolutionary change. The four DNA bases, adenine (A), thymine (T), guanine (G), and cytosine (C) interact with each other and form the hydrogen bonds in the form of A and T; G and C (purine and pyrimidine interaction) in the middle of the double-helix strands. The base pairs are the building blocks of DNA strands that can be further transcribed and translated to make essential proteins in human body.
What is DNA sequencing?
DNA sequencing is a laboratory method developed early in 1977 to determine the precise sequence of nucleotides or bases that make up the DNA strands which is very important in many fields such as forensic science, biotechnology and archaeology. Maxam and Gilbert developed a chemical cleavage method of radiolabelled DNA sequencing using gamma-32P (toxic chemical) along with the same time with Sanger. Frederick Sanger introduced the enzymic method (chain-termination sequencing method) by using dideoynucleotides (ddNTPs; ddATPs, ddTTPs, ddGTPs and ddCTPs) which function as chain terminators. This method is modified by Smith et.al. in 1986 to run the electrophoresis in one lane instead of four by adding a fluorescent dye to the 5’end of a primer. The fluorescence-based DNA sequencing is very effective as it is an automated and rapid process to produce a chromatogram of the order of DNA molecules. There are two different chain termination methods that involved fluorescent tags in the DNA sequencing method: the dye primer technique and the dye terminator method. The fluorescently labelled dideoxynucleotides at the 3’ end with four different dyes are called fluorochromes that functioned as a chain terminator which will emit light at different wavelengths when it passed through the laser beam. On the other hand, the dye primer technique tagged a primer at the 5’end by using four different colour dyes called fluorophore groups with different emission maxima wavelength that formed the bands on the gel (2).
Figure 1 Comparison of the output between Sanger sequencing method that produced autoradiogram (LEFT) while fluorescence-based sequencing method generated chromatogram (RIGHT). Copy right from google
What is the quantum-based material or bio-technology you will be talking about?
What does it do, on a general level?
Structure
The fluorescence-based DNA sequencing is a chain termination approach associated with the polymerase chain reaction (PCR). For the dye termination method, different lengths of the DNA fragments are generated with the fluorochrome (ddNTPs) randomly incorporated with the DNA fragments at the 3’end of DNA complementary strands. Enzymes such as DNA polymerase and primer (oligonucleotide) are used to synthesize multiple DNA complementary strands and undergo further elongation until fluorochrome is attached. The circular plasmid is denatured at 96 degree Celsius and allow the primer to attach on the template strand which catalyzed by a DNA polymerase. Besides that, the DNA polymerase also helped to catalyzed the polymerisation of dNTPs from the direction of 5’ to 3’ by adding deoxynucleotide triphosphates (dNTPs) until chain terminator (ddNTPs) is added. Dideoxynucleotides blocked the elongation of the DNA strand due to the absence of hydroxyl group at the 3’ end. The DNA strand is denatured again and the processes are repeated to produce multiple different length DNA fragments. The DNA fragments are then run on a polyacrylamide gel tube to perform capillary electrophoresis where electrostatic force moved the DNA fragments across the gel. The negatively charged phosphate group of the DNA molecules are repelled from the negative pole and moved toward positive pole. The separation of fragments by lengths as the smaller size fragments moved faster across the gel when the current is switched on. The sequence was passed through a laser beam then a highly sensitive fluorescence detector (spectroscopy) to read the bands. The electropherogram generated shown the sequence of bases of the complementary strand. Figure 2 showed the brief process on the DNA sequencing of the dye termination method (3). Contradictorily, the dye primer method run the labelled primer DNA into four different colour sequencing reactions with the association of each colour to one base (dNTPs and ddNTPs) (2). For example, the red labelled primer is reacted in the isolated container together with the dATPs and ddATPs to produce variable length of DNA complementary stands. The fluorescently dyed primer bound to the 5’end of the DNA strands that allowed the elongation of complementary strand until the chain terminator was added. The four reaction mixtures are combined and run together in single lane onto the slab gel for electrophoresis (4). The bands of the fluorescent tags on the primers were detected automatically which are consistent to the four different bases.
Figure 2 This is the flowchart of the dye termination process of the fluorescence-based DNA sequencing method (3).
What is the overall chemical / biochemical / material structure of your specific system?
How is your system synthesized/fabricated/biosynthesized?
What are the key chemical components required for its function?
Function
There are different sets of dye used for DNA sequencing but the introduction of ET dyes (two dyes per primer, one functioned as a donor and another acted as acceptor) optimized the energy absorption and minimized the electrophoretic shifts (2). By detecting the emission wavelength of the bands produced using the fluorescence resonance energy transfer (FRET), the types of the bases can be efficiently determined. FRET allowed the energy of the laser beam to transfer to the acceptor dye after absorbed by a donor molecule which then only emits light at the characteristic wavelength for detection and determination of the specific terminator (5). The four fluorescent tags attached have different wavelength of absorption at a common emission wavelength (480nm) when the laser beam strike onto it. The four spectrally distinct fluorescent dyes used have the emission maximum wavelength range from 500nm to 610nm (6). The donor fluorophores are electronically excited by the argon laser beam and transfer the excitation energy to the neighbour chromophore acceptor through the non-radioactive excitation of long-range dipole-dipole intermolecular coupling (7). Figure 3 illustrated the flow of energy from donor molecule to acceptor molecule that produce a better quantum efficiency and narrower bandwidth (8). This is a non-radioactive quantum mechanical process that does not involve the collision between molecules due to the similar resonance frequency that lead to the secondary emission light has longer wavelength and lower energy.
Figure 3 The illustration of the fluorescence resonance energy transfer (FRET) with a diagram (8).
What are the fundamental quantum principles of operation of your system? Give diagrams as appropriate.
Applications
DNA sequencing techniques are used to analyse genomes and the encoded proteins to identify the alternation of genes associated with diseases and phenotypes. Besides that, the recognition genotype of the proteins provides a guidance to potential drug targeting. DNA sequencing technologies are the fundamental methods in biological fields and can be used in different areas to determine the genomes of certain species for further utilization. Decoding DNA helped to increase the understanding of organism at different level as the DNA carry the genetic information that controlled most of the cellular activity in the organism. In molecular biology, DNA sequencing is used to decode the genomes of the organism and the information collected is linked to the study of encoding protein by using codons. The chain of polypeptides is formed (primary protein structure) and it can undergo further interaction with other polypeptides to form secondary, tertiary or quaternary protein structure. The functions of those proteins synthesized can be analysed as the distinct chemical properties amino acids linked are known with the method of DNA sequencing. Furthermore, alteration of genomes can be examined generation by generation by using DNA sequencing to study about evolutionary biology and the phenomena of evolution. The DNA sequencing technologies also used to classify the species in the diversity into taxonomic groups according to their DNA sequences. For examples, the DNA of human and chimpanzee have shared 98.8% of similarities after comparison using DNA sequencing and supported the theory of the human evolution from the lineage of chimpanzee. Moreover, DNA sequencing method can aid the food safety management by regulating the invasion of pathogen as the identification and characterisation of microorganisms is fast to prevent diseases outbreak (9). In addition, DNA sequencing may be utilized for the forensic identification by sequencing and comparing the DNA to the DNA profiles stored in the databases. Also, paternity testing as there is no 100% identical DNA or fingerprints presence in the world and hence the percentage of similarities indicated the family relationships. Finally, DNA sequencing method also very important in medicine industry to detect the presence of inherited disease or cancers such as Huntington disease, Tay-Sachs and sickle cell anaemia as the alteration or mutation of gene bases will adversely affect the functions of the proteins. Genetic Alliance, District of Columbia Department of Health (10) stated that family history played an essential role as a powerful diagnostic tool to provide the potential health problems that might occur.
How is your system used – for what specific applications?
Why are these applications important?
Future
What are the main weaknesses of the technology you have investigated?
The fluorescent-based DNA sequencing also known as Sanger method is one of the very first generation of DNA sequencing and have some drawbacks as the cost per run is very expensive and the process is time consuming. The fluorescence-based DNA sequencing only used to determine the order of nucleotides of one DNA strand with the dideoxynucleotides (ddNTPs) were permanently labelled. According to Miodrag (11), the disadvantage of the fluorescence-based sequencing method is the small mononucleotides involved were not nicely separated and hard to analyse using computer due to its size and hence decreases the accuracy of the reading. Besides that, the high temperature used during denaturation of the DNA strands might cause undesirable damage to the sequences as DNA molecules are linked covalently. The 1st generation sequencing method have been modified and innovated to create a better and cost-effective methods. Nowadays, the DNA sequencing techniques and methodology has been evolved to next generation sequencing with greater performance to enhance the efficiency and accuracy of the system to decode the genes. Figure 4 showed the timeline and reading capacity of the DNA sequencing techniques developed after Sanger method (12). For instance: –
2nd generation 454 Sequencing- Pyrosequencing that involve pyrophosphate as an initiator of the reactions and produced a stronger light signal; Illumina platform that performed reversible termination sequencing with high output; SOLiD technology sequenced by annellation and ligation of oligonucleotides that have high accuracy of sequencing (12). Besides that, quantum mechanics theory approached to DNA sequencing that helped to decrease the instrumental and operational cost with a simple and portable personalize genome sequencing machines. For example, ion torrent technology is the first non-optical sensors to detect the bases of DNA molecules that use semiconductor based hydrogen detection system (13). Moreover, the development of nanopore tunnelling current DNA sequencing which introduced label-free detection by identifying the fluctuation of electrical signal when the molecules passing through a nanometer-sized holes in a biological membrane (14).
Figure 4 The timeline of the next generation DNA sequencing techniques (12)
How can this technology be improved in future? Or evolve / be used for bio-inspired technology development (for biological systems)?