Sequencing

Sequencing refers to the biotechnology techniques that determine the order of the genetic material. The genetic material that acts as the blueprint for most cells and organisms is deoxyribonucleic acid (DNA) . DNA provides the information to make ribonucleic acid (RNA) , which in turn provides the information to produce protein.

The information for all living things is stored in the genetic material that is part of the organism . An apt analogy is that of a book containing information in the form of letters that make up words. When interpreted by reading and comprehending, the letters on the book's pages take on an order. Likewise, an organism's genetic material is a sequence of chemical letters. Without some interpretation, this information is useless. Prokaryotic organisms such as bacteria and more complex, multicellular organisms such as humans have built in systems that determine the information that the genetic material conveys. These systems function to determine the order of the information, or the sequence in which the information is presented.

Humans have also learned to decipher the genetic code by sequencing techniques. As well, the identification and arrangement of the components that make up proteins (amino acids) can be determined by other sequencing techniques.

Knowing the sequence of the genetic material has allowed scientists to determine what stretches of the material might specify proteins, or to detect alterations in the genetic material that might be important in genetic diseases, such as cystic fibrosis or cancer . As well, sequence information allows researchers to specifically change the arrangement of the genetic material (a mutation ), in order to determine if the mutation affects the functioning of the cell or organism.

Knowledge of the protein sequence allows researchers to use powerful computers and computer software to study the three-dimensional structure of the protein molecule and to assess how mutations in the protein sequence affect the shape and function of the protein. Also, the shape of a protein is important in designing chemicals like antibiotics that will specifically target the protein and bind to it.

DNA sequencing determines the order of the compounds that make up the DNA. These compounds are called bases. There are four bases; adenine, thymine, guanine and cytosine.

Beginning in the early 1990s and culminating about a decade later, the best-known example of sequencing has been the effort to sequence the human genome . The human genome is the genetic material that is carried in human cells.

In the laboratory, the sequencing of DNA is done by allowing the manufacture of DNA to begin and then stopping the process in a controlled way (i.e., at a certain base at a known location in the DNA). This can be accomplished by two methods. The first method is called the Sanger-Coulson procedure, after its two creators. In the procedure, a small amount of what is termed a dideoxynucleoside base is mixed into the solution that contains the four regular bases. A dideoxynucleoside base is slightly different in structure from the normal base and is also radioactively labeled. When the radioactive base is added on to the growing DNA chain, the next regular base cannot be attached to it. Thus, lengthening of the DNA stops. By using four different dideoxynucleotides that are structurally different from the four regular bases, a pattern of DNA interruption occurs as a number of experiments are done. This produces DNA pieces of many different lengths that have all begun from the same start point. The different pieces can be visualized using the technique of gel electrophoresis , and the jig-saw puzzle pattern of different lengths can be sorted out to deduce the base sequence of the original DNA.

The second DNA sequencing technique is known as the Maxam-Gilbert technique, once again after the scientists who pioneered the technique. Here, both strands of the double-stranded DNA are labeled using radioactive phosphorus (phosphorus is an element that is makes up part of the four bases of DNA). The DNA is heated, which causes the two strands to separate from one another. Both strands are then cut up into a number of shorter pieces using specific enzymes. The differently sized fragments of each DNA strand can be separated using gel electrophoresis, and the resulting patterns determine the sequence of each DNA strand.

The Sanger-Coulson method has been modified so as to be done using automated DNA sequencing machines. This enables DNA to be sequenced much faster than is possible manually.

A sequencing method called shotgun sequencing was successfully used as one approach to sequence the human genome. In shotgun sequencing, the use of a variety of enzymes that cut DNA at different and specific sites produces hundreds or thousands of random bits. Each small stretch of DNA is automatically sequenced and then powerful computers piece back together the information to generate the entire DNA genome sequence.

Protein sequencing determines the arrangement of the amino acids of the protein. This can be done indirectly if the DNA sequence is known. From that sequence, the RNA sequence can be deduced, followed by the sequence of amino acids that the RNA codes for. If the DNA sequence is not known, then the protein sequence can be determined directly, using a chemical approach. The most popular chemical sequencing technique is the Edman degradation procedure. The amino acids are chemically snipped off one at a time from one end of a protein. Each released amino acid can be identified using a technique called reverse phase chromatography . By keeping the identified amino acids in order, the sequence of the protein is determined.

Another protein sequencing technique is called fast atom bombardment mass spectrometry (FAB-MS). Here, the sample is bombarded with a stream of quickly moving atoms . Typically, argon atoms are used. The interaction of the atoms with the protein causes the protein to become charged. When the protein is chemically broken into fragments the charged regions can be used to identify the amino acids. FAB-MS is a powerful technique, although highly specialized and expensive equipment is required.

Another more widely used protein sequencing technique employs a variety of protein degrading enzymes to break up a protein into fragments. The shorter fragments, which are called peptides, can then be sequenced. The enzymes that are used cut the protein into fragments in an overlapping manner. That is, an end of one fragment will have the same information as the end of another fragment. These areas of common information allows researchers to piece the sequence back together to reveal the amino acid arrangement in the intact protein.

See also DNA synthesis; DNA technology.

Resources

books

Alphey, L. DNA Sequencing: From Experimental Methods to Bioinformatics. Berlin: Springer-Verlag, 1997.

Graham, C.A., and A.J.M. Hill. DNA Sequencing Protocols. 2nd ed. Clifton, NJ: Humana Press, 2001.

Kinter, M., and N.E. Sherman. Protein Sequencing and Identification Using Tandem Mass Spectroscopy. Hoboken, NJ: Wiley-Interscience, 2000.

Smith, B.J. Protein Sequencing Protocols (Methods in Molecular Biology, V. 211. Clifton, NJ: Humana Press, 2002.

Brian Hoyle

Sequencing

Resources

Sequencing refers to the biotechnology techniques that determine the order of the genetic material. The genetic material that acts as the blueprint for most cells and organisms is deoxyribonucleic acid (DNA). DNA provides the information to make ribonucleic acid (RNA), which in turn provides the information to produce protein. Both DNA and RNA can be sequenced. Protein can also be sequenced to determine the order of amino acids that makes up the molecule.

The information for all living things is stored in the genetic material that is part of the organism. An apt analogy is that of a book containing information in the form of letters that make up words. When interpreted by reading and comprehending, the letters on the book’s pages take on an order. Likewise, an organism’s genetic material is a sequence of chemical letters. Without some interpretation, this information is useless. Prokaryotic organisms such as bacteria and more complex, multicellular organisms such as humans have built in systems that determine the information that the genetic material conveys. These systems function to determine the order of the information, or the sequence in which the information is presented.

Humans have also learned to decipher the genetic code by sequencing techniques. As well, the identification and arrangement of the components that make up proteins (amino acids) can be determined by other sequencing techniques.

Knowing the sequence of the genetic material has allowed scientists to determine what stretches of the material might specify proteins, or to detect alterations in the genetic material that might be important in genetic diseases, such as cystic fibrosis or cancer. As well, sequence information allows researchers to specifically change the arrangement of the genetic material (a mutation), in order to determine if the mutation affects the functioning of the cell or organism.

Knowledge of the protein sequence allows researchers to use powerful computers and computer software to study the three-dimensional structure of the protein molecule and to assess how mutations in the protein sequence affect the shape and function of the protein. Also, the shape of a protein is important in designing chemicals like antibiotics that will specifically target the protein and bind to it.

DNA sequencing determines the order of the compounds that make up the DNA. These compounds are called bases. There are four bases in DNA; adenine, thymine, guanine, and cytosine.

Beginning in the early 1990s and culminating about a decade later, the best-known example of sequencing has been the effort to sequence the human genome. The human genome is the genetic material that is carried in human cells.

In the laboratory, the sequencing of DNA is done by allowing the manufacture of DNA to begin and then stopping the process in a controlled way (i.e., at a certain base at a known location in the DNA). This can be accomplished by two methods. The first method is called the Sanger-Coulson procedure, after its two creators. In the procedure, a small amount of what is termed a dideoxynucleoside base is mixed into the solution that contains the four regular bases. A dideoxynucleoside base is slightly different in structure from the normal base and is also radioactively labeled. When the radioactive base is added on to the growing DNA chain, the next regular base cannot be attached to it. Thus, lengthening of the DNA stops. By using four different dideoxynucleotides that are structurally different from the four regular bases, a pattern of DNA interruption occurs as a number of experiments are done. This produces DNA pieces of many different lengths that have all begun from the same start point. The different pieces can be visualized using the technique of gel electrophoresis, and the jig-saw puzzle pattern of different lengths can be sorted out to deduce the base sequence of the original DNA.

The second DNA sequencing technique is known as the Maxam-Gilbert technique, once again after the scientists who pioneered the technique. Here, both strands of the double-stranded DNA are labeled using radioactive phosphorus (phosphorus is an element that makes up part of the four bases of DNA). The DNA is heated, which causes the two strands to separate from one another. Both strands are then cut up into a number of shorter pieces using specific enzymes. The differently sized fragments of each DNA strand can be separated using gel electrophoresis, and the resulting patterns determine the sequence of each DNA strand.

The Sanger-Coulson method has been modified so as to be done using automated DNA sequencing machines. This enables DNA to be sequenced much faster than is possible manually.

A sequencing method called shotgun sequencing was successfully used as one approach to sequence the human genome. In shotgun sequencing, the use of a variety of enzymes that cut DNA at different and specific sites produces hundreds or thousands of random bits. Each small stretch of DNA is automatically sequenced and then powerful computers piece back together the information to generate the entire DNA genome sequence.

Protein sequencing determines the arrangement of the amino acids of the protein. This can be done indirectly if the DNA sequence is known. From that sequence, the RNA sequence can be deduced, followed by the sequence of amino acids that the RNA codes for. If the DNA sequence is not known, then the protein sequence can be determined directly, using a chemical approach. The most popular chemical sequencing technique is the Edman degradation procedure. The amino acids are chemically snipped off one at a time from one end of a protein. Each released amino acid can be identified using a technique called reverse phase chro-matography. By keeping the identified amino acids in order, the sequence of the protein is determined.

Another protein sequencing technique is called fast atom bombardment mass spectrometry (FAB-MS). Here, the sample is bombarded with a stream of quickly moving atoms. Typically, argon atoms are used. The interaction of the atoms with the protein causes the protein to become charged. When the protein is chemically broken into fragments the charged regions can be used to identify the amino acids. FAB-MS is a powerful technique, although highly specialized and expensive equipment is required.

Another more widely used protein sequencing technique employs a variety of protein degrading enzymes to break up a protein into fragments. The shorter fragments, which are called peptides, can then be sequenced. The enzymes that are used cut the protein into fragments in an overlapping manner. That is, an end of one fragment will have the same information as the end of another fragment. These areas of common information allows researchers to piece the sequence back together to reveal the amino acid arrangement in the intact protein.

Resources

BOOKS

Brown, Terry. Gene Cloning and DNA Analysis: An Introduction. Boston: Blackwell Publishing, 2006.

Primrose, Sandy B., Richard M. Twyman, and Robert W. Old. Principles of Gene Manipulation. Boston: Blackwell Publishing, 2002.

Zimmer, Marc. Glowing Genes: A Revolution In Biotechnology. Loughton, UK: Prometheus Books, 2005.

Brian Hoyle

Sequencing

█ BRIAN HOYLE

Sequencing refers to the techniques used to determine the order of the constituent bases (i.e., adenine, thymine, guanine, and cytosine) of deoxyribonucleic acid (DNA) or protein. Protein sequencing determines the order of the constituent amino acids. Sequencing is increasingly important in forensic science and in the rapid and positive identification of potential pathogens that can be exploited by bioterrorists.

DNA is typically sequenced for several reasons: to determine the sequence of the protein encoded by the DNA, the location of sites at which restriction enzymes can cut the DNA, the location of DNA sequence elements that regulate the production of messenger RNA, or alterations in the DNA.

The sequencing of DNA is accomplished by stopping the lengthening of a DNA chain at a known base and at a known location in the DNA. Practically, this can be done in two ways. In the first method, called the Sanger-Coulson procedure, a small amount of a specific so-called dideoxynucleoside base is incorporated in along with a mixture of the other four normal bases. This base is slightly different from the normal base and is radioactively labeled. The radioactive base becomes incorporated into the growing DNA chain instead of the normal base, growth of the DNA stops. This stoppage is done four times, each time using one of the four different dideoxynucleosides. This generates four collections of DNA molecule. Also, because replication of the DNA always begins at the same point, and because the amount of altered base added is low, for each reaction many DNA pieces of different length will be generated. When the sample is used for gel electrophoresis, the different sized pieces can be resolved as radioactive bands in the gel. Then, with the location of the bases known, the sequence of the DNA can be deduced. The second DNA sequencing technique is known as the Maxam-Gilbert technique, after its co-discoverers. In this technique, both strands of double-stranded DNA are radioactively labeled using radioactive phosphorus. Upon heating, the DNA strands separate and can be physically distinguished from each other, as one strand is heavier than the other. Both strands are then cut up using specific enzymes, and the different sized fragments of DNA are separated by gel electrophoresis. Based on the pattern of fragments the DNA sequence is determined.

The Sanger-Coulsom is the more popular method. Various modifications have been developed and it has been automated for very large-scale sequencing. During the sequencing of the human genome, a sequencing method called shotgun sequencing was very successfully employed. Shotgun sequencing refers to a method that uses enzymes to cut DNA into hundreds or thousands of random bits. So many fragments are necessary since automated sequencing machines can only decipher relatively short fragments of DNA about 500 bases long. The many sequences are then pieced back together using computers to generate the entire DNA genome sequence.

Protein sequencing involves determining the arrangement of the amino acid building blocks of the protein. It is common to sequence a protein by the DNA sequence encoding the protein. This, however, is only possible if a cloned gene is available. It still is often the case that chemical protein sequencing, as described subsequently, must be performed in order to manufacture an oligonucleotide probe that can then be used to locate the target gene. The most popular direct protein chemical sequencing technique in use today is the Edman degradation procedure. This is a series of chemical reactions, that remove one amino acid at a time from a certain end of the protein (the amino terminus). Each amino acid that is released has been chemically modified in the release reaction, allowing the released product to be detected using a technique called reverse phase chromatography. The identity of the released amino acids is sequentially determined, producing the amino acid sequence of the protein.

Another protein sequencing technique is called fast atom bombardment mass spectrometry, or FAB-MS. This is a powerful technique in which the sample is bombarded with a stream of fast atoms, such as argon. The protein becomes charged and fragmented in a sequence-specific manner. The fragments can be detected and their identify determined. The expense and relative scarcity of the necessary equipment can be a limitation to the technique.

Still another protein sequencing strategy is the digestion of the protein with specialized protein-degrading enzymes called proteases. The shorter fragments that are generated, called peptides, can then be sequenced. The problem then is to order the peptides. This is done by the use of two proteases that cut the protein at different points, generating overlapping peptides. The peptides are separated and sequenced, and the patterns of overlap and the resulting protein sequence can be deduced.

█ FURTHER READING:

BOOKS:

Cirincione, Joseph, Jon B. Wolfsthal, Miriam Rajkuman, Jessica T. Mathews. Deadly Arsenals: Tracking Weapons of Mass Destruction. Washington, DC: Carnegie Endowment for International Peace, 2002.

PERIODICALS:

Balding D. J. "The DNA Database Search Controversy." Biometrics 2002 Mar; 58 (1): 241–4.

Henderson J. P. "The Use of DNA Statistics in Criminal

Trials." Forensic Sci Int. 2002 Aug 28; 128 (3): 183–6.

Mullis, K. B. and F. A. Faloona."Specific Synthesis of DNA in vitro via a Polymerase catalysed Chain Reaction."Methods in Enzymology no. 155 (1987): 335–50.

SEE ALSO

Anthrax Weaponization
Biological Weapons, Genetic Identification
Genetic Information: Ethics, Privacy and Security Issues
Genetic Technology
Genomics
Infectious Disease, Threats to Security

Sequencing

Molecular techniques of analysis are a vital part of forensic science . Analysis of genetic material is instrumental in detecting pathogenic (disease-causing) microorganisms and in identifying a victim or implicating a suspect.

These molecular examinations rely on the determining of the arrangement of the building blocks of the genetic material. This determination is called sequencing.

Sequencing refers to the techniques used to determine the order of the constituent bases (i.e., adenine, thymine, guanine, and cytosine) of deoxyribonucleic acid (DNA ), ribonucleic acid (RNA) or the constituent amino acid building blocks of protein.

DNA is typically sequenced for several reasons: to determine the sequence of the protein encoded by the DNA, the location of sites at which restriction enzymes can cut the DNA, the location of DNA sequence elements that regulate the production of messenger RNA, or to detect alterations in the DNA.

The sequencing of DNA is accomplished by stopping the lengthening of a DNA chain at a known base and at a known location in the DNA. Practically, this can be done in two ways. In the first method, called the Sanger-Coulson procedure, a small amount of a specific so-called dideoxynucleoside base is incorporated in a mixture with the other four normal bases. This base is slightly different from the normal base and is radioactively labeled. The radioactive base becomes incorporated into the growing DNA chain instead of the normal base, and growth of the DNA stops. This stoppage is done four times, each time using one of the four different dideoxynucleosides. This generates four collections of DNA molecules. Also, because replication of the DNA always begins at the same point, and because the amount of altered base added is low, for each reaction many DNA pieces of different lengths will be generated. When the sample is used for gel electrophoresis , the different-sized pieces can be resolved as radioactive bands in the gel. Then, knowing the location of the bases, the sequence of the DNA can be deduced.

The second DNA sequencing technique is known as the Maxam-Gilbert technique, after its co-discoverers. In this technique, both strands of double-stranded DNA are radioactively labeled using radioactive phosphorus. Upon heating, the DNA strands separate and can be physically distinguished from each other, as one strand is heavier than the other. Both strands are then cut up using specific enzymes and the different sized fragments of DNA are separated by gel electrophoresis. Based on the pattern of fragments, the DNA sequence is determined.

Several decades ago, sequencing involved scrutinizing the gels by eye. However, the marriage of powerful computational hardware and software to the sequencing process has automated the procedure.

The Sanger-Coulsom approach is the more popular method. Various modifications have been developed, and it has been automated for very large-scale sequencing. During the sequencing of the human genome, a sequencing method called shotgun sequencing was very successfully employed. Shotgun sequencing refers to a method that uses enzymes to cut DNA into hundreds or thousands of random bits. So many fragments are necessary since automated sequencing machines can only decipher relatively short fragments of DNA about 500 bases long. The many sequences are then pieced back together using computers to generate the entire DNA genome sequence.

Protein sequencing involves determining the arrangement of the amino acid building blocks of the protein. It is common to sequence a protein by determining the DNA sequence encoding the protein . This, however, is only possible if a cloned gene is available. It is still often the case that chemical protein sequencing, as described subsequently, must be performed in order to manufacture an oligonucleotide probe that can then be used to locate the target gene. The most popular direct protein chemical sequencing technique in use today is the Edman degradation procedure. This is a series of chemical reactions, which remove one amino acid at a time from a certain end of the protein (the amino terminus). Each amino acid that is released has been chemically modified in the release reaction, allowing the released product to be detected using a technique called reverse phase chromatography . The identity of the released amino acids is sequentially determined, producing the amino acid sequence of the protein.

Another protein sequencing technique is called fast atom bombardment mass spectrometry, or FABMS. This is a powerful technique in which the sample is bombarded with a stream of fast atoms, such as argon. The protein becomes charged and fragmented in a sequence-specific manner. The fragments can be detected and their identity determined. The expense and relative scarcity of the necessary equipment can be a limitation to the technique, though.

Still another protein sequencing strategy is the digestion of the protein with specialized protein-degrading enzymes called proteases. The shorter fragments that are generated, called peptides, can then be sequenced. The problem then is to order the peptides. This is done by the use of two proteases that cut the protein at different points, generating overlapping peptides. The peptides are separated and sequenced, and the patterns of overlap and the resulting protein sequence can be deduced.

see also Analytical instrumentation; DNA sequences, unique; PCR (polymerase chain reaction).

sequencing
1. The procedure by which ordered units of data (octets or messages) are numbered, transmitted over a communications network (which may rearrange their order), and reassembled into the original order at their destination.

2. Proceeding through a program in its ordinary order, normally from sequential memory locations. See also loop.