DNA sequencing is the process of determining the precise order of nucleotides in a DNA molecule. It was discovered in the 1970s by Sanger and Gilbert and involves using chain terminating dideoxynucleotides or chemical cleavage to break DNA into fragments of known length. These fragments are then separated by gel electrophoresis and used to deduce the DNA sequence. Automated sequencing uses fluorescent labels instead of radioactivity and allows for high throughput sequencing of many DNA samples at once. DNA sequencing has applications in medicine, forensics, and agriculture.
DNA sequencing is the process of determining the precise order of nucleotides in a DNA molecule. It was discovered in the 1970s by Sanger and Gilbert and involves using chain terminating dideoxynucleotides or chemical cleavage to break DNA into fragments of known length. These fragments are then separated by gel electrophoresis and used to deduce the DNA sequence. Automated sequencing uses fluorescent labels instead of radioactivity and allows for high throughput sequencing of many DNA samples at once. DNA sequencing has applications in medicine, forensics, and agriculture.
DNA sequencing is the process of determining the precise order of nucleotides in a DNA molecule. It was discovered in the 1970s by Sanger and Gilbert and involves using chain terminating dideoxynucleotides or chemical cleavage to break DNA into fragments of known length. These fragments are then separated by gel electrophoresis and used to deduce the DNA sequence. Automated sequencing uses fluorescent labels instead of radioactivity and allows for high throughput sequencing of many DNA samples at once. DNA sequencing has applications in medicine, forensics, and agriculture.
Adm no-9PBG/16 Dept. Of PBG, CA, BBSR, OUAT SUMMARY
What is DNA sequencing
Who and when discovered How it is prepared Its relevant to biological science How long will it give benefits DNA DNA is the molecule that is the hereditary material in all living cells. Genes are made of DNA. A gene consists of enough DNA to code for one protein, and a genome is simply the sum total of an organism's DNA. What is DNA made of? DNA is a very large molecule, made up of smaller units called nucleotides that are strung together in a row, making a DNA molecule thousands of times longer than it is wide. Each nucleotide has three parts: a sugar molecule, a phosphate molecule, and a structure called a nitrogenous base. The nitrogenous base is the part of the nucleotide that carries genetic information. The bases found in DNA come in four varieties: Adenine(A), Cytosine(C), Guanine(G), and Thymine(T). DNA Sequencing DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases A,T,G &C in a strand of DNA. Purpose:- HISTORY:- DNA Friedrich Miescher
James Watson Francis Crick
double-helix Rosalind Franklin Frederick Sanger,
sequencing of proteins the sequencing of
DNA
Frederick Sanger insulin.
bacteriophage φX174 DNA sequencing methods Maxam & Gilbert method
By A.M. Maxam and W.Gilbert-1977
Chemical sequencing Treatment of DNA with certain chemicals- DNA cuts into fragments- monitoring of sequences Procedure 1. Denature a double-stranded DNA to single-stranded by increasing temperature. Radioactively label one 5' end of the DNA fragment to be sequenced by a kinase reaction using gamma-32P. 2. Cleave DNA strand at specific positions using chemical reactions. For example, The method developed by Maxam and Gilbert uses formic acid (fire ant venom) to break DNA after both A and G, dimethyl sulfate (toxic) to break after G, and hydrazine (rocket fuel) to break after C and T or, if you added salt, only after C. 3. The chemical treatments outlined in Maxam-Gilbert's paper cleaved at G, A+G, C and C+T. A+G means that it cleaves at A, but occasionally at G as well. 4. Now in four reaction tubes, we will have several differently sized DNA strands. Fragments are electrophoresed in high-resolution acrylamide gels for size separation. 5. These gels are placed under X-ray film, which then yields a series of dark bands which show the location of radiolabeled DNA molecules. The fragments are ordered by size and so we can deduce the sequence of the DNA molecule. An example Maxam–Gilbert sequencing reaction. Cleaving the same tagged segment of DNA at different points yields tagged fragments of different sizes. The fragments may then be separated by gel electrophoresis. Sanger Method (Enzymatic) Sanger method The chain termination reaction Dideoxynucleotide triphosphatase (ddNTPs) chain terminators {Having an H on the 3’C of the ribose sugar (normally OH found in dNTPs)}
COMPARISON SANGER METHOD MAXAM-GILBERT METHOD Enzymatic Chemical
Requires DNA synthesis Requires DNA
Termination of chain Breaks DNA at different
elongation nucleotides Automation Automation is not available Single-stranded DNA Double stranded or single stranded DNA Automated DNA Sequencing Automated DNA Sequencing is based on the Sanger-Coulson method, with two notable differences from the standard procedure. The first difference concerns the labelling of the products of Polymerase Chain Reaction: automated produce use fluorescent labels in the place of radioactive labelling used in the standard procedure. The fluorescent labels are usually attached to the four dideoxynucleotides used for chain termination. In the four track system of automated DNA Sequencing, each of the four dideoxynucleotides used in a separate reaction, and the products are run in 4 adjacent lanes of the gel. If a different fluorochrome is attached to each of the four dideoxynucleotides, all of them could be used in the same reaction in place of preparing a separate reaction for each dideoxynucleotide. This is called the single track system since the reaction products are run in a single gel lane or capillary. Generally, the DNA to be sequenced is subjected to thermal cycle sequencing to generate the chain terminated polynucleotides required for sequencing. The reaction products are subjected to polyacrylamide gel electrophoresis under denaturing conditions or loaded into a capillary filled with a sequencing gel. The bands produced in the polyacrylamide gel or capillary are identified with the help of a fluorescence detector, which identifies the fluorescent signal emitted by each band. The flurochromes are excited by laser beam and the resulting fluorescence signal in sensed by a photovoltaic cell. The resulting data are fed into a computer, which, in turn, converts these signals into the base sequence of the DNA molecule. The sequence information could be printed out or stored in a data storage device for future use; this is the second major deviation from the standard Sanger- Coulson procedure. In the fourth track system, the sequence can be recognised from the raw data but it has to be interpreted using an appropriate computer program in the single track system; this becomes necessary because the shifts in mobility due to the different fluorochromes have to be compensated for. Automated DNA sequencers can read upto 96 DNA sequences in a 2 hours period, which is extremely fast as compared to manual DNA Sequencing. Automated DNA Sequencing has the following advantages over manual DNA Sequencing:(1) Radioactivity is not used,(2) Gel processing after electrophoresis and autoradiography are not needed,(3) The tedious manual reading of gels is not required as data are processed in a computer, (4) The sequence data is directly fed into and stored in a computer,(5) The separation of the same reaction products can be repeated to recheck the results in cases of doubt since they can be stored for a long period of time and (6) It is extremely fast. Applications of DNA Sequencing Forensics;- to help identify individuals because each individual has a different genetic sequence. . Medicine;- can be used to help detect the genes which are linked to various genetic disorders such as muscular dystrophy. . Agriculture;- The mapping and sequencing of a genome of microorganisms has helped to make them useful for crops and food plants. .Advantages- .1) Improved diagnosis of disease .2) Bio pesticides .3) Identifying crime suspects .4) Sequencing the whole Genome of organisms (e.g. Human genome project) . Disadvantages- .1) Whole genome cannot be sequenced at once .2) Very slow and time consuming