Applications of DNA Sequencing
1. DNA sequencing information is important for planning the procedure and method of gene manipulation.
2. DNA sequencing is used for construction of restriction endonuclease map.
3. DNA sequencing information is used to find tandem repeats or inverted repeat for the possibility of hairpin formations.
4. The sequences can be used to find whether any open reading frame (ORF) coding for a polypeptide exists.
5. DNA sequences can be used to find a polypeptide sequence from the data bank or to compare with DNA sequences from other animals for phylogenetic analysis.
6. DNA sequencing information are used to construct the molecular evolution map.
7. DNA sequencing information are useful in identifying exons and introns.
Friday, October 30, 2009
Application of DNA sequencing -1
The most obvious application of DNA sequencing technology is the accurate sequencing of genes and genomes. Only about 500-800 bases can be sequenced in one experiment so larger DNA molecules, including whole genomes, must be broken into smaller fragments before sequencing and then reassembled by searching for overlaps. Accuracy is achieved by sequencing each template several times.
Lower-fidelity single-pass DNA sequencing is useful for the rapid accumulation of sequence data at the expense of some accuracy. Another application of DNA sequencing technology is resequencing the same DNA molecule over and over. DNA sequencing is most frequently used to determine genome sequence. An example of this type of DNA sequencing application is the human genome project. DNA sequencing its impacts on biomedical research.
Several genes have been identied to associate with genetic conditions, including familial breast cancer and colorectal cancer, Alzheimer's disease, myotonic dystrophy, neurofibromatosis and fragile X syndrome. Ultimately, DNA sequencing will become a part of a patient's medical record, helping physicians to determine the patient's risk of certain diseases and the optimal treatments.
Many emerging science and technological fields rely heavily on DNA sequencing. For instance, nutrigenetics is the study of how our genetic DNA sequencing makeup affects our responses to diet. Nutrigenetics plays a central role in explaining the connection between red meat and colorectal cancer. It was shown that not everyone but only those with specific genotypes are at high risk for colon cancer when consuming large quantities of red meat. The results showed that the extracted genomic DNA can be used for high scale genotyping and genotyping of adaptive and ecological relevant genes.
Lower-fidelity single-pass DNA sequencing is useful for the rapid accumulation of sequence data at the expense of some accuracy. Another application of DNA sequencing technology is resequencing the same DNA molecule over and over. DNA sequencing is most frequently used to determine genome sequence. An example of this type of DNA sequencing application is the human genome project. DNA sequencing its impacts on biomedical research.
Several genes have been identied to associate with genetic conditions, including familial breast cancer and colorectal cancer, Alzheimer's disease, myotonic dystrophy, neurofibromatosis and fragile X syndrome. Ultimately, DNA sequencing will become a part of a patient's medical record, helping physicians to determine the patient's risk of certain diseases and the optimal treatments.
Many emerging science and technological fields rely heavily on DNA sequencing. For instance, nutrigenetics is the study of how our genetic DNA sequencing makeup affects our responses to diet. Nutrigenetics plays a central role in explaining the connection between red meat and colorectal cancer. It was shown that not everyone but only those with specific genotypes are at high risk for colon cancer when consuming large quantities of red meat. The results showed that the extracted genomic DNA can be used for high scale genotyping and genotyping of adaptive and ecological relevant genes.
Details on how DNA Sequencing works
DNA sequencing reactions are just like the PCR reactions for replicating DNA (refer to the previous page DNA Denaturation, Annealing and Replication). The reaction mix includes the template DNA, free nucleotides, an enzyme (usually a variant of Taq polymerase) and a 'primer' - a small piece of single-stranded DNA about 20-30 nt long that can hybridize to one strand of the template DNA.
Automated sequencing gel: That's exactly what we do to sequence DNA, then - we run DNA replication reactions in a test tube, but in the presence of trace amounts of all four of the dideoxy terminator nucleotides. Electrophoresis is used to separate the resulting fragments by size and we can 'read' the sequence from it.
A DNA molecule carries information in the form of four chemical groups or bases, represented by the letters A, C, G and T. The order of bases on a DNA strand is the DNA sequence.
Most DNA sequencing is carried out using the chain termination method. This involves the synthesis of new DNA strands on a single stranded template and the random incorporation of chain-terminating nucleotide analogues.
The chain termination method produces a set of DNA molecules differing in length by one nucleotide. The last base in each molecule can be identified by way of a unique label. Separation of these DNA molecules according to size places them in the correct order to read off the sequence.
Sequencing is achieved by including in each reaction a nucleotide analogue that cannot be extended and thus acts as a chain terminator. Four reactions are set up, each containing the same template and primer but a chain terminator specific for A, C, G or T. Because only a small amount of the chain terminator is included, incorporation into the new DNA strand is a random event. Each reaction therefore generates a collection of fragments, but every DNA strand will end at the same type of base (A, C, G or T).
The primers or nucleotides included in each of the four reactions contain different fluorescent labels allowing DNA strands terminating at each of the four bases to be identified. The reaction products are then mixed and separated by gel electrophoresis, which separates DNA molecules according to size even if they differ in length by only a single nucleotide. As the DNA strands pass a specific point, the fluorescent signal is detected and the base identified. The whole process can be extensively automated.
Automated sequencing gel: That's exactly what we do to sequence DNA, then - we run DNA replication reactions in a test tube, but in the presence of trace amounts of all four of the dideoxy terminator nucleotides. Electrophoresis is used to separate the resulting fragments by size and we can 'read' the sequence from it.
A DNA molecule carries information in the form of four chemical groups or bases, represented by the letters A, C, G and T. The order of bases on a DNA strand is the DNA sequence.
Most DNA sequencing is carried out using the chain termination method. This involves the synthesis of new DNA strands on a single stranded template and the random incorporation of chain-terminating nucleotide analogues.
The chain termination method produces a set of DNA molecules differing in length by one nucleotide. The last base in each molecule can be identified by way of a unique label. Separation of these DNA molecules according to size places them in the correct order to read off the sequence.
Sequencing is achieved by including in each reaction a nucleotide analogue that cannot be extended and thus acts as a chain terminator. Four reactions are set up, each containing the same template and primer but a chain terminator specific for A, C, G or T. Because only a small amount of the chain terminator is included, incorporation into the new DNA strand is a random event. Each reaction therefore generates a collection of fragments, but every DNA strand will end at the same type of base (A, C, G or T).
The primers or nucleotides included in each of the four reactions contain different fluorescent labels allowing DNA strands terminating at each of the four bases to be identified. The reaction products are then mixed and separated by gel electrophoresis, which separates DNA molecules according to size even if they differ in length by only a single nucleotide. As the DNA strands pass a specific point, the fluorescent signal is detected and the base identified. The whole process can be extensively automated.
DNA Sequencing method
Shotgun DNA Sequencing method-Shotgun DNA sequencing is a method for determining the sequence fo a very large piece of DNA. The basic DNA sequencing reaction can only get the sequence of a few hundred nucleotides. For larger ones (like BAC DNA), we usually fragment the DNA and insert the resultant pieces into a convenient vector replicate them. After we sequence the fragments, we try to deduce from them the sequence of the original BAC DNA.Shotgun DNA sequencing: assembly of random sequence fragments. To sequence a BAC, we take millions of copies of it and chop them all up randomly. We then insert those into plasmids and for each one we get, we grow lots of it in bacteria and sequence the insert. If we do this to enough fragments, eventually we'll be able to reconstruct the sequence of the original BAC based on the overlapping fragments we've sequenced.
The most commonly used method of sequencing DNA - the dideoxy or chain termination method was developed by Fred Sanger in 1977 (for which he won his second Nobel Prize). The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, it stops the replication reaction.
The chain termination method produces a set of DNA molecules differing in length by one nucleotide. The last base in each molecule can be identified by way of a unique label. Separation of these DNA sequencing molecules according to size places them in the correct order to read off the dna sequencing.
The most commonly used method of sequencing DNA - the dideoxy or chain termination method was developed by Fred Sanger in 1977 (for which he won his second Nobel Prize). The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, it stops the replication reaction.
The chain termination method produces a set of DNA molecules differing in length by one nucleotide. The last base in each molecule can be identified by way of a unique label. Separation of these DNA sequencing molecules according to size places them in the correct order to read off the dna sequencing.
Introduction to DNA Sequencing
What is DNA, and how it determine our characteristics?DNA is basically a long molecule that contains coded instructions for the cells. Everything the cells do is coded somehow in DNA - which cells should grow and when, which cells should die and when, which cells should make hair and what color it should be. Our DNA is inherited from our parents. We resemble our parents simply because our bodies were formed using DNA to guide the process - the DNA we inherited from them.
First we must learn how DNA is structured. DNA is a long molecule, like a chain, where the links of the chain are pieces called nucleotides . There are four different types of nucleotides in DNA which we'll call 'A', 'G', 'C' and 'T'. These four are all that's necessary to write a code that describes our entire body plan.
DNA chains are made by connecting those nucleotides together via chemical bonds. At right is a diagram showing four nucleotides connected to form an oligonucleotide. Double-stranded DNA is simply two chains of single- stranded DNA, positioned so their "bases" can interact with each other. At left is a cartoon depiction of double-stranded DNA.
The bases in the middle "pair up" with bases on the opposite strand, so that a type 'A' nucleotide is always opposite a type 'T', and 'G' is opposite 'C'. The attraction between the paired nucleotides is fairly weak, but when there is a whole string of them, it adds up to enough strength to hold the strands together.
First we must learn how DNA is structured. DNA is a long molecule, like a chain, where the links of the chain are pieces called nucleotides . There are four different types of nucleotides in DNA which we'll call 'A', 'G', 'C' and 'T'. These four are all that's necessary to write a code that describes our entire body plan.
DNA chains are made by connecting those nucleotides together via chemical bonds. At right is a diagram showing four nucleotides connected to form an oligonucleotide. Double-stranded DNA is simply two chains of single- stranded DNA, positioned so their "bases" can interact with each other. At left is a cartoon depiction of double-stranded DNA.
The bases in the middle "pair up" with bases on the opposite strand, so that a type 'A' nucleotide is always opposite a type 'T', and 'G' is opposite 'C'. The attraction between the paired nucleotides is fairly weak, but when there is a whole string of them, it adds up to enough strength to hold the strands together.
DNA Sequencing Technology
DNA sequencing is the process of determining the exact order of the bases A, T, C and G in a piece of DNA. In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA.DNA is basically a long molecule that contains coded instructions for the cells. Everything the cells do is coded somehow in DNA - which cells should grow and when, which cells should die and when, which cells should make hair and what color it should be. Our DNA is inherited from our parents. We resemble our parents simply because our bodies were formed using DNA to guide the process - the DNA we inherited from them.
The most obvious application of DNA sequencing technology is the accurate sequencing of genes and genomes. Only about 500-800 bases can be sequenced in one experiment so larger DNA molecules, including whole genomes, must be broken into smaller fragments before sequencing and then reassembled by searching for overlaps. Accuracy is achieved by sequencing each template several times.
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