IT is a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase, are used to amplify the number of copies of a target DNA sequence by more than 100 times in a few hours. American molecular biologist Kary Mullis developed the techniques of PCR in the 1970s. For his ingenious invention, he was awarded the 1993 Nobel Prize in physiology or medicine.
PCR amplification of DNA is like any DNA replication by DNA polymerase in vivo. (in lving cells) The difference is that PCR produces DNA in a test tube. For a PCR reaction to proceed, four components are necessary: template, primer, deoxyribonecleotides (adenine, thymine, cytosine, guanine) and DNA polymerase. In addition, part of the sequence of the targeted DNA has to be known in order to design the according primers. In the first step, the targeted double stranded DNA is heated to over 194°F (90°C) for denaturation. During this process, two strands of the targeted DNA are separated from each other. Each strand is capable of being a template. The second step is carried out around 122° (50°C). At this lowered temperature, the two primers anneal to their complementary sequence on each template. The DNA polymerase then extends the primer using the provided NUCLEOTIDES. As a result, at the end of each cycle, the numbers of DNA molecules double.
PCR was initially carried out manually in incubators of different temperatures for each step until the extraction of DNA polymerase from thermophilic bacteria. The bacterium Thermus aquaticus was found in Yellow Stone National Park. This bacterium lives in the hot springs at 203°F (95°C). The DNA polymerase from T. aquaticus keeps its activity at above 95°C for many hours. Several additional heat-resistant DNA polymerases have also now been identified.
Sunday, February 28, 2010
DNA Fingerprinting
Genetic, genomic, or DNA fingerprinting is the term applied to a range of techniques that are used to show similarities and dissimilarities between the DNA present in different individuals.
Genetic fingerprinting is an important tool in the arsenal of forensic investigators. Genetic fingerprinting allows for positive identification, not only of body remains, but also of suspects in custody. Genetic fingerprinting can also link suspects to physical evidence.
Sir Alec Jeffreys at the University of Leicester developed DNA fingerprinting in the mid 1980s. The sequence of NUCLEOTIDES in DNA is similar to a fingerprint, in that it is unique to each person. DNA fingerprinting is used for identifying people, studying populations, and forensic investigations.
Genetic fingerprinting is an important tool in the arsenal of forensic investigators. Genetic fingerprinting allows for positive identification, not only of body remains, but also of suspects in custody. Genetic fingerprinting can also link suspects to physical evidence.
Sir Alec Jeffreys at the University of Leicester developed DNA fingerprinting in the mid 1980s. The sequence of NUCLEOTIDES in DNA is similar to a fingerprint, in that it is unique to each person. DNA fingerprinting is used for identifying people, studying populations, and forensic investigations.
Recombinant DNA
Deoxyribonucleic acid (DNA) is the information blueprint that exists in most living organisms. Some viruses instead contain ribonucleic acid (RNA). Even these viruses require the production of DNA at some stage of their replication.
DNA from different organisms of the same species combines together naturally to yield an organism that has traits from both parent organisms. There is also evidence accumulating that DNA transfer between different species may be a natural process. However, much interspecies mixing of DNA is the result of deliberate experimental manipulations.
A crucial process of these manipulations is the preparation of recombinant DNA. Recombinant DNA is DNA from different organisms that have been chemically bonded together to form a single DNA. The recombinant DNA can be interpreted by the various enzymes of prokaryotic or eukaryotic cells, so that the genes contained in the recombinant DNA can be expressed and the protein products produced.
The recombination can involve the DNA from two eukaryotic organisms, two prokaryotic organisms, or between an eukaryote and a prokaryote. An example of the latter is the production of human insulin by the bacterium E .COLI, which has been achieved by splicing the gene for insulin into the E. coli genome such that the insulin gene is expressed and the protein product formed.
DNA from different organisms of the same species combines together naturally to yield an organism that has traits from both parent organisms. There is also evidence accumulating that DNA transfer between different species may be a natural process. However, much interspecies mixing of DNA is the result of deliberate experimental manipulations.
A crucial process of these manipulations is the preparation of recombinant DNA. Recombinant DNA is DNA from different organisms that have been chemically bonded together to form a single DNA. The recombinant DNA can be interpreted by the various enzymes of prokaryotic or eukaryotic cells, so that the genes contained in the recombinant DNA can be expressed and the protein products produced.
The recombination can involve the DNA from two eukaryotic organisms, two prokaryotic organisms, or between an eukaryote and a prokaryote. An example of the latter is the production of human insulin by the bacterium E .COLI, which has been achieved by splicing the gene for insulin into the E. coli genome such that the insulin gene is expressed and the protein product formed.
Molecular Biology
Molecular biology is an interdisciplinary approach to understanding biological functions and regulation at the level of molecules such as nucleic acids, proteins, and carbohydrates Following the rapid advances in biological science brought about by the development and advancement of the Watson-Crick model of DNA (deoxyribonucleic acid) during the 1950s and 1960s, molecular biologists studied gene structure and function in increasing detail. In addition to advances in understanding genetic machinery and its regulation, molecular biologists continue to make fundamental and powerful discoveries regarding the structure and function of cells and of the mechanisms of genetic transmission. The continued study of these processes by molecular biologists and the advancement of molecular biological techniques requires integration of knowledge derived from physics, chemistry, mathematics, genetics, biochemistry, cell biology and other scientific fields.
Molecular biology also involves organic chemistry, physics , and biophysical chemistry as it deals with the physicochemical structure of macromolecules (nucleic acids, proteins, lipids, and carbohydrates) and their interactions. Genetic materials including DNA in most of the living forms orRNA (ribonucleic acid) in all plant viruses and in some animal viruses remain the subjects of intense study.
In 1945, William Astbury coined the term "molecular biology" referring to the study of the chemical and physical structure of biological macromolecules (large sized molecules). There was and still is a strong belief that all forms of life have uniformity in biological processes. The pioneer findings in prokaryotes (a simple or primitive cell type, e.g., bacteria and blue green alga) are extended to eukaryotes (a complex or well developed cell type, e.g., animal and plant cells).
The complete set of instructions for making an organism (i.e., the complete set of genes) is called its genome. It contains the master blueprint for all cellular structures and activities for the lifetime of the cell or organism. The human genome consists of tightly coiled threads of deoxyribonucleic acid (DNA) and associated protein molecules organized into structures called chromosomes. In humans, as in other higher organisms, a DNA molecule consists of two strands that wrap around each other to resemble a twisted ladder whose sides, made of sugar and phosphate molecules, are connected by rungs of nitrogen-containing chemicals called bases. Each strand is a linear arrangement of repeating similar units called nucleotides, which are each composed of one sugar, one phosphate, and a nitrogenous base. Four different bases are present in DNA adenine (A), thymine (T), cytosine (C), and guanine (G). The particular order of the bases arranged along the sugar-phosphate backbone is called the DNA sequence; the sequence specifies the exact genetic instructions required to create a particular organism with its own unique traits.
Each time a cell divides into two daughter cells, its full genome is duplicated; for humans and other complex organisms, this duplication occurs in the nucleus. During cell division the DNA molecule unwinds and the weak bonds between the base pairs break, allowing the strands to separate. Each strand directs the synthesis of a complementary new strand, with free nucleotides matching up with their complementary bases on each of the separated strands. Strict base-pairing rules are adhered to. Adenine will pair only with thymine (an A-T pair) and cytosine with guanine (a C-G pair). Each daughter cell receives one old and one new DNA strand. The cells adherence to these base-pairing rules ensures that the new strand is an exact copy of the old one. This minimizes the incidence of errors (mutations) that may greatly affect the resulting organism or its offspring.
Each DNA molecule contains many genes, the basic physical and functional units of heredity. A gene is a specific sequence of nucleotide bases, whose sequences carry the information required for constructing proteins, which provide the structural components of cells and tissues as well as enzymes for essential biochemical reactions.
The central dogma of molecular biology is that DNA is copied to make mRNA (messenger RNA) and mRNA is used as the template to make proteins. Formation of RNA is called transcription and formation of protein is called translation. Transcription and translation processes are regulated at various stages and the regulation steps are unique to prokaryotes and eukaryotes. DNA regulation determines what type and amount of mRNA should be transcribed, and this subsequently determines the type and amount of protein. This process is the bottom line for growth and morphogenesis.
Molecular biology also involves organic chemistry, physics , and biophysical chemistry as it deals with the physicochemical structure of macromolecules (nucleic acids, proteins, lipids, and carbohydrates) and their interactions. Genetic materials including DNA in most of the living forms orRNA (ribonucleic acid) in all plant viruses and in some animal viruses remain the subjects of intense study.
In 1945, William Astbury coined the term "molecular biology" referring to the study of the chemical and physical structure of biological macromolecules (large sized molecules). There was and still is a strong belief that all forms of life have uniformity in biological processes. The pioneer findings in prokaryotes (a simple or primitive cell type, e.g., bacteria and blue green alga) are extended to eukaryotes (a complex or well developed cell type, e.g., animal and plant cells).
The complete set of instructions for making an organism (i.e., the complete set of genes) is called its genome. It contains the master blueprint for all cellular structures and activities for the lifetime of the cell or organism. The human genome consists of tightly coiled threads of deoxyribonucleic acid (DNA) and associated protein molecules organized into structures called chromosomes. In humans, as in other higher organisms, a DNA molecule consists of two strands that wrap around each other to resemble a twisted ladder whose sides, made of sugar and phosphate molecules, are connected by rungs of nitrogen-containing chemicals called bases. Each strand is a linear arrangement of repeating similar units called nucleotides, which are each composed of one sugar, one phosphate, and a nitrogenous base. Four different bases are present in DNA adenine (A), thymine (T), cytosine (C), and guanine (G). The particular order of the bases arranged along the sugar-phosphate backbone is called the DNA sequence; the sequence specifies the exact genetic instructions required to create a particular organism with its own unique traits.
Each time a cell divides into two daughter cells, its full genome is duplicated; for humans and other complex organisms, this duplication occurs in the nucleus. During cell division the DNA molecule unwinds and the weak bonds between the base pairs break, allowing the strands to separate. Each strand directs the synthesis of a complementary new strand, with free nucleotides matching up with their complementary bases on each of the separated strands. Strict base-pairing rules are adhered to. Adenine will pair only with thymine (an A-T pair) and cytosine with guanine (a C-G pair). Each daughter cell receives one old and one new DNA strand. The cells adherence to these base-pairing rules ensures that the new strand is an exact copy of the old one. This minimizes the incidence of errors (mutations) that may greatly affect the resulting organism or its offspring.
Each DNA molecule contains many genes, the basic physical and functional units of heredity. A gene is a specific sequence of nucleotide bases, whose sequences carry the information required for constructing proteins, which provide the structural components of cells and tissues as well as enzymes for essential biochemical reactions.
The central dogma of molecular biology is that DNA is copied to make mRNA (messenger RNA) and mRNA is used as the template to make proteins. Formation of RNA is called transcription and formation of protein is called translation. Transcription and translation processes are regulated at various stages and the regulation steps are unique to prokaryotes and eukaryotes. DNA regulation determines what type and amount of mRNA should be transcribed, and this subsequently determines the type and amount of protein. This process is the bottom line for growth and morphogenesis.
DNA Microarray Technology
An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots.
Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene.
Types of Microarrays:
Depending upon the kind of immobilized sample used construct arrays and the information fetched, the Microarray experiments can be categorized in three ways:
1. Microarray expression analysis: In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease.
2. Microarray for mutation analysis: For this analysis, the researchers use gDNA. The genes might differ from each other by as less as a single nucleotide base. A single base difference between two sequences is known as Single Nucleotide Polymorphism (SNP) and detecting them is known as SNP detection
3. Comparative Genomic Hybridization: It is used for the identification in the increase or decrease of the important chromosomal fragments harboring genes involved in a disease.
Applications of Microarrays:
Gene discovery: DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions.
Disease diagnosis: DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.
Drug discovery: Microarray technology has extensive application in Pharmacogenomics.Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
Toxicological research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants.
Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene.
Types of Microarrays:
Depending upon the kind of immobilized sample used construct arrays and the information fetched, the Microarray experiments can be categorized in three ways:
1. Microarray expression analysis: In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease.
2. Microarray for mutation analysis: For this analysis, the researchers use gDNA. The genes might differ from each other by as less as a single nucleotide base. A single base difference between two sequences is known as Single Nucleotide Polymorphism (SNP) and detecting them is known as SNP detection
3. Comparative Genomic Hybridization: It is used for the identification in the increase or decrease of the important chromosomal fragments harboring genes involved in a disease.
Applications of Microarrays:
Gene discovery: DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions.
Disease diagnosis: DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.
Drug discovery: Microarray technology has extensive application in Pharmacogenomics.Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
Toxicological research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants.
DNA Replication – The Replisome
The replisome is a complex molecular machine that carries out replication of DNA. It is comprised of a number of subcomponents, each performing a specific function during the process of replication. Helicase is an enzyme which breaks the hydrogen bonds between the two strands of DNA, thus separating the strands ahead of DNA synthesis. As helicase unwinds the double helix, it induces the formation of supercoils in other areas of the DNA.
Gyrase relaxes and undoes the supercoiling which has been caused by the helicase by cutting the DNA strands, allowing it to rotate and release the supercoil, and then rejoining the strands. Gyrase is most commonly located upstreak of the replication fork -- where the supercoils are being formed.
Primase adds complementary RNA primers to a DNA strand to begin Okazaki fragments. In addition, because DNA Polymerae can only continue (but not begin) a strand, Primase begins the leading strand as well.
DNA polymerase III is comprised of two catalytic cores -- one for replication of the leading strand and one for the lagging strand. DNA polemerase III, however, cannot stay on the DNA strand long enough to efficiently replicate a daughter strand. Hence, DNA polymerase III stays on the strands via a dimer beta clamp which contains three subunits that come together to enclose the strand -- ensuring that DNA polymerase III will remain on the strand for a few thousand nucleotides as opposed to a few hundred.
DNA polymerase I removes the RNA primers set by Primase and completes the Okazaki fragments. Because there is such a small gap remaining after the action of DNA polymerase I has continued the strand of the Okazaki fragment, ligase is required to fill in the gap. The two ends of the Okazaki fragments are subsequently connected by covalent bonds.
Single-strand binding proteins bind to the exposed bases in an effort to counteract their instability and prevent the single-strand DNA from hydrogen-bonding to itself to form dangerous hairpin structures.
DNA polymerases contain a ‘proofreading’ mechanism, commonly referred to as ‘exonuclease activity’. This removes nucleotides that have been mistakenly added.
DNA Replication – Signature of Design
DNA Replication stands as a fundamental challenge to those who seek to hold to a Darwinian worldview. As the process by which biological information is copied and passed on to succeeding generations, the mechanism is fundamental to the process of self-replication of cells. Yet self-replication of cells is necessary for the operation of any selective process such as natural selection. Thus, to attempt to explain the immense sophistication of this mechanism with reference to natural selection requires one to presuppose the existence of the very thing they wish to explain. Because of its extremely sophisticated nature, most biochemists previously reckoned that the system arose once, prior to the origin of the last universal common ancestor. In addition, many biochemists have long regarded the close functional similarity of DNA replication observed in all life as evidence for the single origin of DNA replication. Yet in 1999 researchers from the National Institutes of Health demonstrated that the core enzymes involved in the DNA replication machinery of bacteria and archae/eukaryotes (the two major trunks of the evolutionary tree of life) did not in fact share a common evolutionary origin. It thus appears as if two identical DNA replication systems have emerged independently in bacteria and archae -- after these two evolutionary lineages supposedly diverged from the last universal common ancestor.
It is phenomenal to think that this engineering marvel evolved a single time, let alone twice. There exists no obvious reason for DNA replication to take place by a semiconservative, RNA primer-dependent, bidirectional mechanism that depends on leading and lagging strands to produce DNA daughter molecules. Even if DNA replication could have evolved independently on two separate occasions, it is reasonable to expect that fundamentally different mechanisms would emerge for bacteria and the archae/eukaryotes given their idiosyncrasies. But, they did not.
Gyrase relaxes and undoes the supercoiling which has been caused by the helicase by cutting the DNA strands, allowing it to rotate and release the supercoil, and then rejoining the strands. Gyrase is most commonly located upstreak of the replication fork -- where the supercoils are being formed.
Primase adds complementary RNA primers to a DNA strand to begin Okazaki fragments. In addition, because DNA Polymerae can only continue (but not begin) a strand, Primase begins the leading strand as well.
DNA polymerase III is comprised of two catalytic cores -- one for replication of the leading strand and one for the lagging strand. DNA polemerase III, however, cannot stay on the DNA strand long enough to efficiently replicate a daughter strand. Hence, DNA polymerase III stays on the strands via a dimer beta clamp which contains three subunits that come together to enclose the strand -- ensuring that DNA polymerase III will remain on the strand for a few thousand nucleotides as opposed to a few hundred.
DNA polymerase I removes the RNA primers set by Primase and completes the Okazaki fragments. Because there is such a small gap remaining after the action of DNA polymerase I has continued the strand of the Okazaki fragment, ligase is required to fill in the gap. The two ends of the Okazaki fragments are subsequently connected by covalent bonds.
Single-strand binding proteins bind to the exposed bases in an effort to counteract their instability and prevent the single-strand DNA from hydrogen-bonding to itself to form dangerous hairpin structures.
DNA polymerases contain a ‘proofreading’ mechanism, commonly referred to as ‘exonuclease activity’. This removes nucleotides that have been mistakenly added.
DNA Replication – Signature of Design
DNA Replication stands as a fundamental challenge to those who seek to hold to a Darwinian worldview. As the process by which biological information is copied and passed on to succeeding generations, the mechanism is fundamental to the process of self-replication of cells. Yet self-replication of cells is necessary for the operation of any selective process such as natural selection. Thus, to attempt to explain the immense sophistication of this mechanism with reference to natural selection requires one to presuppose the existence of the very thing they wish to explain. Because of its extremely sophisticated nature, most biochemists previously reckoned that the system arose once, prior to the origin of the last universal common ancestor. In addition, many biochemists have long regarded the close functional similarity of DNA replication observed in all life as evidence for the single origin of DNA replication. Yet in 1999 researchers from the National Institutes of Health demonstrated that the core enzymes involved in the DNA replication machinery of bacteria and archae/eukaryotes (the two major trunks of the evolutionary tree of life) did not in fact share a common evolutionary origin. It thus appears as if two identical DNA replication systems have emerged independently in bacteria and archae -- after these two evolutionary lineages supposedly diverged from the last universal common ancestor.
It is phenomenal to think that this engineering marvel evolved a single time, let alone twice. There exists no obvious reason for DNA replication to take place by a semiconservative, RNA primer-dependent, bidirectional mechanism that depends on leading and lagging strands to produce DNA daughter molecules. Even if DNA replication could have evolved independently on two separate occasions, it is reasonable to expect that fundamentally different mechanisms would emerge for bacteria and the archae/eukaryotes given their idiosyncrasies. But, they did not.
Therapeutic Cloning
Somatic cell nuclear transfer can also be used to create a clonal embryo. The most likely scenario for this is to produce embryos for use in research, particularly stem cell research. This process is also called "research cloning" or "therapeutic cloning."
Therapeutic cloning is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.
Therapeutic cloning is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.
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