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molecular tools restriction enzyme for recombinant DNA in a biotechnology revolution
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molecular tools restriction enzyme for recombinant DNA in a biotechnology revolution



Introduction
Watson and Crick's description, in 1953, of the double helical structure of the DNA molecule (See Classic Collection- The Structure of DNA) opened the door to a new era in biological understanding and research. Scientists, now knowing the molecular structure of the hereditary molecule, could begin both to elucidate and to manipulate its function. These new studies were, however, dependent on the discovery and use of the many enzymes that are able to modify or join existing DNA molecules, or to aid in the synthesis of new DNA molecules.
DNA enzymology
The late 1950's and the decade of the 60's saw enormous breakthroughs in DNA enzymology. For example, it was during this time that Arthur Kornberg and colleagues isolated DNA polymerase (1955), B. Weiss and C.C. Richardson isolated DNA ligase (1966), and H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized the first sequence specific restriction nuclease (1968). These enzymes, respectively, play roles in the synthesis of DNA molecules, the attachment of two or more DNA molecules to one another, and the breaking of DNA molecules into fragments. Importantly, these enzymes make it possible to create entirely new kinds of DNA molecules and, equally important, to manipulate the functioning of the genes located on these new molecules.
Phage growth restriction
It had been known since the 1950's that phage particles that grow well and efficiently infect one strain of bacteria are often unable to grow well and infect other strains of the same bacterial species. In addition, phage particles that do succeed in infecting a second strain often show the opposite pattern: they are able to efficiently infect the second strain while growing only poorly in the original strain. A series of studies showed that phage particles that efficiently grow and infect host cells have DNA molecules that have been chemically modified by the addition of methyl groups to some of their adenine and/or cytosine bases, while the DNA of poorly infecting phage particles does not show this pattern of chemical modification or "methylation." Phage particles with unmethylated DNA do not grow and infect efficiently because their DNA molecules are cleaved and degraded by enzymes of the host cell, while methylated DNA is protected from this degradation. This phenomenon of degrading unmethylated DNA destroys the growth ability of the phage, and is responsible for the pattern of growth restriction described above.
Methylase and nuclease
In the late 1960's, scientists Stewart Linn and Werner Arber isolated examples of the two types of enzymes responsible for phage growth restriction in Escherichia coli (E. coli) bacteria. One of these enzymes methylated DNA, while the other cleaved unmethylated DNA at a wide variety of locations along the length of the molecule. The first type of enzyme was called a "methylase" while the other was called a "restriction nuclease." These enzymatic tools were important to scientists who were gathering the tools needed to "cut and paste" DNA molecules. What was needed now was a tool that would cut DNA at specific sites, rather than at random sites along the length of the molecule, so that scientists could cut DNA molecules in a predictable and reproducible way.
Site-specific nuclease
This important development came when H.O. Smith, K.W. Wilcox, and T.J. Kelley, working at Johns Hopkins University in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence. Working with Haemophilus influenzae bacteria, this group isolated an enzyme, called HindII, that always cut DNA molecules at a particular point within a specific sequence of six base pairs. This sequence is:

5' G T ( pyrimidine: T or C) ( purine: A or G) A C 3'
3' C A ( purine: A or G) ( pyrimidine: T or C) T G 5'

They found that the HindII enzyme always cuts directly in the center of this sequence. Wherever this particular sequence of six base pairs occurs unmodified in a DNA molecule, HindII will cleave both DNA backbones between the 3rd and 4th base pairs of the sequence. Moreover, HindII will only cleave a DNA molecule at this particular site. For this reason, this specific base sequence is known as the "recognition sequence" for HindII.

HindII is only one example of the class of enzymes known as restriction nucleases. In fact, more than 900 restriction enzymes, some sequence specific and some not, have been isolated from over 230 strains of bacteria since the initial discovery of HindII. These restriction enzymes generally have names that reflect their origin--The first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated. For example EcoRI comes from Escherichia coli RY13 bacteria, while HindII comes from Haemophilus influenzae strain Rd. Numbers following the nuclease names indicate the order in which the enzymes were isolated from single strains of bacteria. Nucleases are further described by addition of the prefix "endo" or "exo" to the name: The term "endonuclease" applies to sequence specific nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule. Nucleases that function by removing nucleotides from the ends of the molecule are called "exonucleases."
Endonucleases and DNA fragments
A restriction endonuclease functions by "scanning" the length of a DNA molecule. Once it encounters its particular specific recognition sequence, it will bond to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones of the double helix. The positions of these two cuts, both in relation to each other, and to the recognition sequence itself, are determined by the identity of the restriction endonuclease used to cleave the molecule in the first place. Different endonucleases yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way, no matter what DNA molecule it is acting on. Once the cuts have been made, the DNA molecule will break into fragments.
Endonucleases and sticky ends
Not all restriction endonucleases cut symmetrically and leave blunt ends like HindII described above. Many endonucleases cleave the DNA backbones in positions that are not directly opposite each other. For example, the nuclease EcoRI has the following recognition sequence:

5' G A A T T C 3'
3' C T T A A G 5'

When the enzyme encounters this sequence, it cleaves each backbone between the G and the closest A base residues. Once the cuts have been made, the resulting fragments are held together only by the relatively weak hydrogen bonds that hold the complementary bases to each other. The weakness of these bonds allows the DNA fragments to separate from one each other. Each resulting fragment has a protruding 5' end composed of unpaired bases. Other enzymes create cuts in the DNA backbone which result in protruding 3' ends. Protruding ends--both 3' and 5'-- are sometimes called "sticky ends" because they tend to bond with complementary sequences of bases. In other words, if an unpaired length of bases (5' A A T T 3') encounters another unpaired length with the sequence (3' T T A A5') they will bond to each other--they are "sticky" for each other. Ligase enzyme is then used to join the phosphate backbones of the two molecules. The cellular origin, or even the species origin, of the sticky ends does not affect their stickiness. Any pair of complementary sequences will tend to bond, even if one of the sequences comes from a length of human DNA, and the other comes from a length of bacterial DNA. In fact, it is this quality of stickiness that allows production of recombinant DNA molecules, molecules which are composed of DNA from different sources, and which has given birth to an industry!

Posted on: 2009/9/9 13:41
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molecular tools: plasmids for recombinant DNA in a biotechnology revolution
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molecular tools: plasmids for recombinant DNA in a biotechnology revolution

Plasmids

A plasmid is an independent, circular, self-replicating DNA molecule that carries only a few genes. The number of plasmids in a cell generally remains constant from generation to generation. Plasmids are autonomous molecules and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using bacteria (see also alkaline lysis) They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.

There are two categories of plasmids. Stringent plasmids replicate only when the chromosome replicates. This is good if you are working with a protein that is lethal to the cell. Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to chromosome.

So how do we manipulate these plasmids?

1. Mutate them using restriction enzymes, ligation enzymes, and PCR. Mutagenesis is easily accomplished by using restriction enzymes to cut out portions of one genome and insert them into a plasmid. PCR can also be used to facilitate mutagenesis. Plasmids are mapped out indicating the locations of their origins of replication and restriction enzyme sites.

2. Select them using genetic markers. Some bacteria are antibiotic resistant. While this is a serious health problem, it is a godsend to molecular scientists. The gene that confers antibiotic resistance can be added (ligated) to the gene you are inserting into the plasmid. So every plasmid that contains your target gene will not be killed by antibiotics. After you transfect your bacterial cells with your engineered plasmid (the one with the target gene and the antibiotic resistant marker), you incubate them in a nutrient broth that also contains antibiotic (usually ampecillin). Any cells that were not transfected (this means they do not have your target gene in them) are killed by the antibiotic. The ones that do have the gene also have the antibiotic resistant gene, and therefore survive the selection process.

3. Isolate them (such as with alkaline lysis)

4. Transform them into cells where they become vectors to transport foreign genes into a recipient organism.

There are some minimum requirements for plasmids that are useful for recombination techniques:

1. Origin of replication (ORI). They must be able to replicate themselves or they are of no practical use as a vector.

2. Selectable marker. They must have a marker so you can select for cells that have your plasmids.

3. Restriction enzyme sites in non-essential regions. You don't want to be cutting your plasmid in necessary regions such as the ORI.

In addition to these necessary requirements, there are some factors that make plasmids either more useful or easier to work with.

1. Small. If they are small, they are easier to isolate (you get more), handle (less shearing), and transform.

2. Multiple restriction enzyme sites. More sites give you greater flexibility in cloning, perhaps even allowing for directional cloning.

3. Multiple ORIs. It is important to note that two genes must have different ORIs if they are going to be inserted in the same plasmid.

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Posted on: 2009/8/27 14:04
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The "central dogma" in biology can be briefly described as follows: "DNA is transcribed to RNA and R
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The "central dogma" in biology can be briefly described as follows: "DNA is transcribed to RNA and RNA is translated to a protein." What happens during transcription? What happens during translation? --- Translation of DNA


DNA Translation

Deoxyribonucleic acid (DNA) is composed of a sequence of nucleotide bases paired together to form a double-stranded helix structure. Through a series of complex biochemical processes the nucleotide sequences in an organism's DNA are translated into the proteins it requires for life. The object of this problem is to write a computer program which accepts a DNA strand and reports the protein generated, if any, from the DNA strand.

The nucleotide bases from which DNA is built are adenine, cytosine, guanine, and thymine (hereafter referred to as A, C, G, and T, respectively). These bases bond together in a chain to form half of a DNA strand. The other half of the DNA strand is a similar chain, but each nucleotide is replaced by its complementary base. The bases A and T are complementary, as are the bases C and G. These two ``half-strands'' of DNA are then bonded by the pairing of the complementary bases to form a strand of DNA.

Typically a DNA strand is listed by simply writing down the bases which form the primary strand (the complementary strand can always be created by writing the complements of the bases in the primary strand). For example, the sequence TACTCGTAATTCACT represents a DNA strand whose complement would be ATGAGCATTAAGTGA. Note that A is always paired with T, and C is always paired with G.

From a primary strand of DNA, a strand of ribonucleic acid (RNA) known as messenger RNA (mRNA for short) is produced in a process known as transcription. The transcribed mRNA is identical to the complementary DNA strand with the exception that thymine is replaced by a nucleotide known as uracil (hereafter referred to as U). For example, the mRNA strand for the DNA in the previous paragraph would be AUGAGCAUUAAGUGA.

It is the sequence of bases in the mRNA which determines the protein that will be synthesized. The bases in the mRNA can be viewed as a collection of codons, each codon having exactly three bases. The codon AUG marks the start of a protein sequence, and any of the codons UAA, UAG, or UGA marks the end of the sequence. The one or more codons between the start and termination codons represent the sequence of amino acids to be synthesized to form a protein. For example, the mRNA codon AGC corresponds to the amino acid serine (Ser), AUU corresponds to isoleucine (Ile), and AAG corresponds to lysine (Lys). So, the protein formed from the example mRNA in the previous paragraph is, in its abbreviated form, Ser-Ile-Lys.

The complete genetic code from which codons are translated into amino acids is shown in the table below (note that only the amino acid abbreviations are shown). It should also be noted that the sequence AUG, which has already been identified as the start sequence, can also correspond to the amino acid methionine (Met). So, the first AUG in a mRNA strand is the start sequence, but subsequent AUG codons are translated normally into the Met amino acid.


http://acm.uva.es/p/v3/385.html

Posted on: 2009/8/17 15:37
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The "central dogma" in biology can be briefly described as follows: "DNA is transcribed to RNA and R
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The "central dogma" in biology can be briefly described as follows: "DNA is transcribed to RNA and RNA is translated to a protein." What happens during transcription? What happens during translation? --- Transcription of DNA

The majority of genes are expressed as the proteins they encode. The process occurs in two steps:

* Transcription = DNA → RNA
* Translation = RNA → protein

Taken together, they make up the "central dogma" of biology: DNA → RNA → protein.


More detail: http://users.rcn.com/jkimball.ma.ultr ... ages/T/Transcription.html

Posted on: 2009/8/10 14:45
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How does DNA replicate? - Polymerase chain reaction in a biotechnology revolution
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How does DNA replicate? - Polymerase chain reaction in a biotechnology revolution

Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a thermostable DNA polymerase. Repeating this process through multiple cycles produces amplification of the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, increasing exponentially

Posted on: 2009/8/3 14:54
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How does DNA replicate? - Rolling circle replication in a biotechnology revolution
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How does DNA replicate? - Rolling circle replication in a biotechnology revolution


Another method of copying DNA, sometimes used in vivo by bacteria and viruses, is the process of rolling circle replication.[16] In this form of replication, a single replication fork progresses around a circular molecule to form multiple linear copies of the DNA sequence. In cells, this process can be used to rapidly synthesize multiple copies of plasmids or viral genomes.

In the cell, rolling circle replication is initiated by an initiator protein encoded by the plasmid or virus DNA. This protein is able to nick one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin (DSO) and remains bound to the 5' phosphate end of the nicked strand. The free 3' hydroxyl end is released and can serve as a primer for DNA synthesis. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Continued DNA synthesis produces multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series. In vivo these linear copies are subsequently converted to double-stranded circular molecules.

Rolling circle replication can also be performed in vitro and has found wide uses in academic research and biotechnology, often used for amplification of DNA from very small amounts of starting material. Replication can be initiated by nicking a double-stranded circular DNA molecule or by hybridizing a primer to a single-stranded circle of DNA. The use of a reverse primer (or random primers) produces hyperbranched rolling circle amplification, resulting in exponential rather than linear growth of the DNA molecule.


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Posted on: 2009/7/27 14:55
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Terminat
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Termination of replication

Termination of replication

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E coli regulate this process through the use of termination sequences which, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.[15]

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular manner. Because eukaryotes have linear chromosomes, DNA replication often fails to synthesize to the very end of the chromosomes (telomeres), resulting in telomere shortening. This is a normal process in somatic cells cells are only able to divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, the enzyme telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation.

Posted on: 2009/7/20 14:39
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Bacteria
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Bacteria

Bacteria

Most bacteria do not go through a well-defined cell cycle and instead continuously copy their DNA; during rapid growth this can result in multiple rounds of replication occurring concurrently.[13] Within E coli, the most well-characterized bacteria, regulation of DNA replication can be achieved through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of ATP to ADP, and the levels of protein DnaA. These all control the process of initiator proteins binding to the origin sequences.

Because E coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by a protein (SeqA) which binds and sequesters the origin sequence; in addition, dnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.[14]

ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication each time the origin is copied the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.

Posted on: 2009/7/13 14:40
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Regulati
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Regulation of replication

Regulation of replication

Eukaryotes

Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication occurs during the S phase (Synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases.[12]

The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells which do not proceed through this checkpoint are quiescent in the "G0" stage and do not replicate their DNA.

Replication of chloroplast and mitochondrial genomes occurs independent of the cell cycle, through the process of D-loop replication.

Posted on: 2009/7/2 13:26
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Dynamics
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How does DNA replicate? - DNA replication within the cell in a biotechnology revolution - Dynamics at the replication fork


Dynamics at the replication fork

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead.[10] This build-up would form a resistance that would eventually halt the progress of the replication fork. DNA topoisomerases are enzymes that solve these physical problems in the coiling of DNA. Topoisomerase I cuts a single backbone on the DNA, enabling the strands to swivel around each other to remove the build-up of twists. Topoisomerase II cuts both backbones, enabling one double-stranded DNA to pass through another, thereby removing knots and entanglements that can form within and between DNA molecules.

Bare single-stranded DNA has a tendency to fold back upon itself and form secondary structures; these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.[11]

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template and thereby assisting with processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double stranded DNA, the sliding clamp undergoes a conformational change which releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.

Posted on: 2009/6/15 18:19
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