1 DNA Sequencing Description of Module Subject Name Paper Name Module Name/Title DNA Sequencing Dr. Vijaya Khader Dr. MC Varadaraj 2 DNA Sequencing 1. Objectives 1. DNA sequencing Introduction 2. Types of methods available 3. Next generation sequencing 2. Lay Out 3 DNA Sequencing Sequencing Maxam Gilbert Sanger's Next generation 3 Introduction 4 DNA Sequencing In the mid 1970s when molecular cloning techniques in general were rapidly improving, simple methods were also developed to determine the nucleotide sequence of DNA. These advances laid the foundation for the detailed analysis of the structure and function of the large number of genes. Other fields which utilize DNA sequencing include diagnostic and forensic biology. With the invention of DNA sequencing, research and discovery of new genes is increased many folds. Using this technique whole genomes of many animal (human), plant, and microbial genomes have been sequenced. The first attempts to sequence DNA mirrored techniques developed in 1960s to sequence RNA. These involved : a) Specific cleavage to the DNA in to smaller fragments by enzymatic digestion or chemical digestion b) Nearest neighbour analysis c) Wandering spot method. Indeed in some studies the DNA was transcribed in to RNA with E.coli RNA polymerase and then sequenced as RNA So there were several questions unanswered; How many base pairs (bp) are there in a human genome?( ~3 billion (haploid)) How much did it cost to sequence the first human genome? ~$2.7 billion How long did it take to sequence the first human genome?( ~13 years) When was the first human genome sequence complete? (2000-2003) Goal figuring the order of nucleotides across a genome Problem Current DNA sequencing methods can handle only short stretches of DNA at once (<1-2Kbp) Solution 5 DNA Sequencing Sequence and then use computers to assemble the small pieces 3.1 Maxam-Gilbert sequencing Also known as chemical sequencing, here there is use radioactive labeling at one 5' end of the DNA fragment which is to be sequenced. This method grew out of studies of the interaction between Lac repressor and lac operator in vitro. In this procedure DNA is radiolabelled at one end is partially cleaved in five different chemical reactions, and these are specific for each base. This generates five polulations of radiolabelled molecules that extend from a common point to the site of terminal cleavage. Each population consists of mixture of different molecules and their length is determined by how the bases are located in DNA. These populations are then resolved by electrophoresis through polyacrlamide gels and the end labelled molecules are detected by autoradiography. This method has not changed in any form or technique in later years, its same as it was devloped. The chemical degradation approach has one advantage over the chain termination sequencing: that the DNA sequence is obtained from the original DNA molecule and it is not an enzymatic copy. Therefore with this method one can sequence synthetic nucleotides, analyze DNA modifications such as DNA methylation and study both DNA secondary structure and interaction of proteins wth DNA be chemical protection or modification interference experiments. 6 DNA Sequencing Figure: Maxam Gilbert Sequencing Method 3.2 Sanger’s Sequencing Frederick Sanger and his colleagues developed the method of sequencing known as chain termination method. This method soon became popular and common method of sequencing used by molecular biologists because of its ease to use and reliability. In this there is less use of toxic chemicals and also less amount of radioactivity is used in comparison to Maxam method. Sanger’s method was automated and it became the commonly used method of DNA sequencing. Next generation sequencing methods are also based on Sanger sequencing method. This method first described the use of: the use of specific primer for extension by DNA polymerase , base specific chain termination, use of polyacrylamide gels to discriminate between single stranded DNA chains differing in length by a single nucleotide. Despite these advances the method was too inaccurate to gain 7 DNA Sequencing general acceptance and it was not until the introduction of chain terminating dideoxynucleoside triphosphates ddNTPs that enzymatic methods of DNA sequencing were used extensively. 2’, 3’ddNTPs differs from conventional DNTPs because of the absence of a hydroxyl residue at the 3’position of deoxyribose. These ddNTPs can be incorporated in to the DNA chain by DNA polymerases by their 5’triphosphate groups. The absence of a 3’hydroxyl residue will not allow to form the phosphodiester bond with the use of DNPT. It leads to prevention of extension of the growing DNA chain . When ddNTP is added with DNTPs, it set in the race between chain extension and termination of chain. The resultant are number of chains of oligonucleotide whose lengths is inferred by the distance between the end of the primer used to start DNA synthesis and the sites of premature termination. In a separate reactions there is use of four separate ddNTPs, number of oligonucleotides are generated which lead to termination at positions of A, C, G or T in the template strand. Steps involved : Mix DNA with dNTPs and ddNTPs Amplify Run in Gel Fragments migrate distance that is proportional to their size 8 DNA Sequencing The 3′-OH group is missing in ddNTPs.which is important for the phosphodiester bond formation labelled primer and template are used in sequencing reaction mixture Dideoxynucleotides are added to all tubes . Fluorescent dyes . Multicyclic molecules which at different wavelengths absorb and emit light. Commonly sued are derivatives of fluorescein and rhodamine. In sequencing, these fluorescent molecules attached to the nucleotides covalently . Separate color is used for four ddNTP. Because we are using a color dye so no need for separate tubes 9 DNA Sequencing Figure: Diagrammatic representation of method 10 DNA Sequencing Figure- Computerized visualization from a single lane of an automated sequencer. Method uses non-radioactive fluorescent labelling. Figure: Chromatogram 11 DNA Sequencing Sanger Sequencing Advantages . Long reads (~900bps) . Suitable for small projects Disadvantages . Low throughput . Expensive 4. Next Generation Sequencing Next-generation sequencing is term which applies to genome sequencing, genome resequencing, transcriptome profiling, protein-DNA interactions. Resequencing is important to study genome variations which is not possible by studying single genome. There is increasing demand for low cost sequencing and this has led to invention of next-generation sequencing or high-throughput sequencing technologies that can produce thousands or millions of sequences simultaneously. NGS is divided further into polony-sequencing based technologies in which there is requirement to amplify DNA before sequencing, and single molecule sequencing which does not require amplification. By this method full E. coli genome was studied in 2005. Applications of NGS include gene expression analysis and re-sequencing. And of course – advancements in chemistry, microscopy and other related technologies enabled the new sequencing technologies. NGS Platforms Roche 454 Illumina Solexa Applied Biosystems SOLiD 12 DNA Sequencing 4.1 454 pyrosequencing In pyrosequencing sequence-based detection technology is used which enables fast and error free quantification of variation in a sequence. It is based on the principle of "sequencing by synthesis." Here chain termination method is not used rather in this technique when there is incorporation of nucleotide there is detection of pyrophosphate release. This technique employs emulsion polymerase chain reaction. DNA is amplified in water droplets in oil emulsion and each droplet contains DNA attached to beads which are coated with primers. The sequencing equipment contains many wells of picoliter-volume with each well containing a single bead and sequencing enzymes. Luciferase enzyme is used in this technique for the generation of light to detect single nucleotides added to nascent DNA. Pooled data is used to generate read outs of sequence. 13 DNA Sequencing 4.2 Illumina (Solexa) sequencing Shankar Balasubramanian and David Klenerman co-founded Solexa in 1988 (it is part of Illumina). Their method of sequencing was based on reversible dye terminator technology and engineered DNA polymerase enzymes. Here primers with DNA template are fixed to slide or flow cell and then amplification is done with polymerase 14 DNA Sequencing which generate clonal DNA colonies or clusters. Four types of reversible terminator bases of four types are used to evaluate the sequence. Image of fluorescent labeled nucleotide is captured by camera. 4.3 SOLiD sequencing Sequencing by ligation technique is being used in SOLiD sequencing. Mixture of oligonucleotides of known and fixed length are labeled according to the sequenced position. The oligonucleotides are first annealed and then ligated. DNA ligase will preferably ligate to matching sequences and will lead to signal corresponding to that nucleotide at that position. Emulsion PCR is used for amplification of DNA prior to sequencing. The resulting 15 DNA Sequencing beads, each of which contain single copies of the similar DNA molecule, are being deposited on a glass slide. This results in sequences of quantities and lengths which are comparable to Illumina sequencing. 16 DNA Sequencing Genome Sequencer 20 (454) Genome analyzer Solexa 4.4 Ion Torrent semiconductor sequencing