Dna sequencing by Dideoxy method
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The document describes the dideoxy method of DNA sequencing. This method was foundational for modern genetic research as it provided the first ability to sequence DNA. It works by using dideoxy nucleotides that lack a hydroxyl group, preventing further nucleotide addition. DNA copies are made with each using one dideoxy nucleotide. After electrophoresis, the sequence is read by the length of DNA fragments terminated at each dideoxy position. While limited to short sequences, it enabled sequencing capabilities and projects like the Human Genome Project.
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The document discusses DNA sequencing techniques, specifically the Sanger method of DNA sequencing. It describes how the Sanger method uses dideoxynucleotides (ddNTPs) which can be incorporated into the growing DNA strand during replication but prevents further elongation. Short primers are used to hybridize and prime DNA synthesis. Microarray analysis is also summarized as measuring the gene expression levels of many genes by isolating mRNA, making cDNA, hybridizing the cDNA to an array chip containing short sequences of many genes.
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DNA sequencing determines the order of nucleotides in DNA. It was first discovered in the late 1800s and early 1900s. In the 1970s, methods for DNA sequencing were developed using primer extension strategies and specific nucleotide labeling. The Maxam-Gilbert method uses purine and pyrimidine specific chemicals to cleave the DNA, while Sanger's dideoxynucleotide method uses chain termination with no 3' OH groups. DNA sequencing is important for fields like molecular medicine, forensics, evolution, and agriculture. It allows detection of bacteria and sequencing of the M13 bacteriophage.
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This document discusses various methods for DNA sequencing, including both early chemical methods and more recent next-generation sequencing technologies. It describes Frederick Sanger's chain termination method from 1977, which uses DNA polymerase, dNTPs, and ddNTPs. It also describes Maxam-Gilbert sequencing from the same year, which uses chemical cleavage. Next-generation methods discussed include pyrosequencing, sequencing by ligation, sequencing by synthesis, and ion semiconductor sequencing. The document compares the different methods based on read length, accuracy, throughput, run time, and cost.
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This document discusses Sanger dideoxy sequencing, which was the first generation of DNA sequencing technologies. It describes the basic structure of nucleotides, how DNA is synthesized, and how dideoxynucleotides are used in the Sanger method. The method involves dividing a DNA sample into four separate sequencing reactions with one dideoxynucleotide each. The reactions are then run on a gel through electrophoresis, separated by size, and detected as fluorescent traces to reveal the DNA sequence.
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1) DNA sequencing refers to determining the order of nucleotide bases (A, G, C, T) in a DNA molecule. This provides essential genetic information for growth and development.
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DNA consists of a linear string of nucleotides, or bases, for simplicity, referred to by the first letters of their chemical names--A, T, C and G. The process of deducing the order of nucleotides in DNA is called DNA sequencing. Since the DNA sequence confers information that the cell uses to make RNA molecules and proteins, establishing the sequence of DNA is key for understanding how genomes work. The technology for DNA sequencing was made faster and less expensive as a part of the Human Genome Project. And recent developments have profoundly increased the efficiency of DNA sequencing even further.
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Dna sequencing by Dideoxy method
- 1. DNA sequencing The dideoxy approach Rajat Patil – 2bv13bt037 PraveenkumarTagare – 2bv13bt032
- 2. Introduction Much of the progress in recent molecular biology and genetic engineering is due to the ability to sequence the DNA. Although large scale efforts of this sort like the Human Genome Project (HGP) used more advanced approaches, the basic DNA sequencing capability arouse from the dideoxy method of DNA sequencing. This method is therefore foundational for most of the research in the field. It can be applied for short sequences only. Large sequences need to be fragmentized.
- 3. • To understand the dideoxy method of DNA sequencing, it is important to know the basic structure of DNA and the nucleotides. • Its made up of three components, • Sugar base – deoxy ribose • Phosphate group • Nitrogenous base ( A,T, G orC) • Accordingly they are called dNTP’s. (dATP, dGTP, dCTP, dTTP) • These lack the hydroxyl group at 3’ carbon atom on the sugar. Deoxy nucleotides
- 5. Dideoxy nucleotides • Its also made up of three components, • Sugar base – Dideoxy ribose • Phosphate group • Nitrogenous base ( A,T, G orC) • Accordingly they are called ddNTP’s. (ddATP, ddGTP, ddCTP, ddTTP) • These lack the hydroxyl group at both 3’ and 4’ carbon atoms on the sugar.
- 7. • In a DNA molecule, each dNTP is bound to the consequent dNTP by a phospho diester bond between 4’ carbon atom of one dNTP and the phosphate group of the next dNTP. • But this is not the case for ddNTPs since they lack the hydroxyl group at 4’ carbon atom of the sugar. • So a chain of DNA terminates when a ddNTP is present.
- 9. • Using this property of a ddNTP, we can sequence a DNA. • Consider a short segment of DNA to be sequenced, - T G ATT C • Four copies of the same sequence are prepared. • It is fed to the PCR and all the necessary enzymes and other chemicals are added. • To each of the copy, three dNTPs are added and one ddNTP is added in equal volumes • 1 - dATP, dGTP, dCTP and (dTTP : ddTTP = 1:1) • 2 - dATP, dTTP, dCTP and (dGTP: ddGTP = 1:1) • 3 - dATP, dTTP, dGTP and (dCTP: ddCTP = 1:1) • 4 - dCTP, dTTP, dGTP and (dATP: ddATP = 1:1) • The four different samples may give rise to many combinations of different sized DNA fragments upon PCR.
- 14. • Now, load these fragments onto a gel and carry out electrophoresis to separate the fragments based on their size.
- 15. • After a while, the bands are obtained.The lighter DNA segments travel furthest and the heavier DNA segments travel the least.
- 16. • To determine the sequence of DNA, we move back from the lightest DNA segment which is a single dideoxy nucleotide. • This chain terminated at the initial point indicating the sequence starts with the dNTP of that ddNTP.
- 17. • Similarly, the next farthest band, which will be a di-nucleotide and the second nucleotide is the dNTP of that ddNTP in which the band was observed.
- 18. • Similarly, all the bands are analyzed and a sequence is created.We cannot predict the last nucleotide because it is not known whether termination occurred because of the ddNTP or not.
- 19. CONCLUSION • This was the first ever method to sequence DNA. • The longer the sequence, the lengthier is the gel required. • Its limitations are overcome by using shotgun sequencing and hierarchal methods for sequencing of the very large genomes of eukaryotes.
- 20. THANKYOU