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DNA SEQUENCE ANALYSIS.pdf
- 2. 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
- 3. WHAT IS A GENE 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)
- 4. 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
- 5. PURPOSE i. Deciphering the code of life ii. Detecting mutations iii. Typing microorganisms iv. Identifying human haplotypes v. Designating polymorphism
- 6. HISTORY ➢ DNA was first discovered and isolated by Friedrich Miescher in 1869 ➢ In 1953, James Watson and Francis Crick put forward their double–helix model of DNA, based on crystallized X-ray structures being studied by Rosalind Franklin ➢ Frederick Sanger, a pioneer of sequencing, Sanger is one of the few scientists who was awarded two Nobel prizes, one for the sequencing of protein, and the other for the sequencing of DNA ➢ The foundation for sequencing proteins was first laid by the work of Frederick Sanger who by 1955 had completed the sequence of all the amino acids in insulin ➢ The first full DNA genome to be sequenced was that of bacteriophage in ΦX174 in 1977 Frederick Sanger
- 7. DNA SEQUENCING METHODS Historically there are two main methods: 1. Maxam and Gilbert method 2. Sanger method 3. Automated sequencing Modern sequencing equipment uses the principles of the sanger technique
- 8. 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
- 9. PROCEDURE 1. Denature double-stranded DNA to single by increasing the temperature Radioactively label one 5’ end of the DNA fragment to be sequenced by a kinase reaction using gamma-32P. 2. Cleave DNA strands 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.
- 10. 3. The chemical treatments outlined in Maxam-Gilbert's paper cleaved at G, A+G, and 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 an X-ray film, which then yields a series of dark bands that show the location of radiolabeled DNA molecules. The fragments are ordered by size so we can deduce the sequence of the DNA molecule.
- 11. An example Maxam-Gilbert sequencing reaction. Cleaving the same tagged segment of DNA at different points fields tagged fragments of different sizes. The fragments may then be separated by gel electrophoresis.
- 12. SANGER METHOD (ENZYMATIC) ➢ A most common approach used for DNA sequencing ➢ Invented by Frederick Sanger-1977 ➢ Nobel prize – 1980 ➢ Also termed as Chain Termination or Dideoxy method
- 13. 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) ➢ssDNA - addition of ddNTPs - elongation stops
- 14. COMPARISON BETWEEN NTP, dNTP, & ddNTP
- 15. PRINCIPLE ssDNA (complementary strand ) Enzymatic synthesis of complementary polynucleotide chains Termination at specific nucleotide positions Separate between Gel/Capillary Electrophoresis Read DNA sequence
- 18. COMPARISON Enzymatic Requires DNA synthesis Termination of chain elongation Automation Single-stranded DNA SANGER METHOD MAXAM-GILBERT METHOD Chemical Requires DNA Breaks DNA at different nucleotides Automation is not available Double-stranded or single- stranded DNA
- 19. 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 labeling of the products of Polymerase Chain Reaction: automated produce use fluorescent labels in place of radioactive labeling 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 is 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.
- 20. ➢ 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 fluorochromes are excited by a laser beam and the resulting fluorescence signal is 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 recognized 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 up to 96 DNA sequences in a 2 hours period, which is extremely fast compared to manual DNA Sequencing,
- 21. Automated DNA Sequencing has the following advantages over manual DNA sequencing: • Radioactivity is not used • Gel processing after electrophoresis and autoradiography is not needed • The tedious manual reading of gels is not required as data is processed in a computer • The sequence data is directly fed into and stored in a computer • 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 • It is extremely fast
- 22. 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 the genome of microorganisms have helped to make them useful for crops and food plants
- 23. ADVANTAGES Improved diagnosis of disease Bio pesticides Identifying crime suspects Sequencing the whole Genome of organisms (e.g. Human genome project)
- 24. DISADVANTAGES Whole genome cannot be sequenced at once Very slow and time-consuming