What is a recombinant DNA organism?

Recombinant Antibody
The term recombinant and especially recombinant DNA regards to an artificial methods of producing DNA (synthetic DNA and proteins).

The manufacturing technique make use of bacterias, these bacterias are genetically modified in such a way that a specific DNA segment (gene) is artificially combined with their original DNA. After this insertion of DNA segment. The bacterias are nourished and kept in ideal conditions for reproduction and after a relative short period of time, after the reproduction, these specific segment of DNA is extracted out of the original DNA bacterias.

In short a segment of DNA is recombined (recombinant) with the original DNA of the bacteria, these genetically modified bacterias are rapidly reproduce and by that reproduce the DNA segment that was recombined with the original genome.

Antibodies are protein molecules produced by our immune system, the shape of an antibody is similar to a Y shape. The role of the antibodies to keep our health is crucial, their task is to recognize and attack foreign bacterias and viruses in our body.

There are many types of antibodies, each type can recognize unique antigen, the antibody Y tips has a unique molecular structure (keys) that combines with the targeted antigen. This attachment of many antibodies to a foreign objects (bacteria or virus) ultimately brings to its destruction.

Using recombinant antibody has significant advantages compared with the conventional antibody and there for its use becoming more popular now days. The fact that no animals are needed in the manufacturing procedure of the recombinant antibodies, in addition, the manufacturing time is relatively short compared with the conventional method. Moreover, the quality of the final product is higher that these of the non recombinant method.

What is cloning? And how did recombinant DNA make cloning possible?

Recombinant DNA

Recombinant DNA technology emerged as a response to the need for specific DNA segments in amounts sufficient for biochemical analysis. The method entails clipping the desired segment out of the surrounding DNA and copying it millions of times. The success of recombinant DNA technology, by which microbial cells can be engineered to produce foreign proteins, relies on the faithful reading of the corresponding genes by bacterial cell machinery, and has fueled most of the recent advances in modern molecular biology. During the last twenty years, studies of cloned DNA sequences have given us a detailed knowledge of gene structure and organization, and have provided clues to the regulatory pathways by which the cell controls gene expression in the multiple cell types comprising the basic vertebrate body plan. Genetic engineering, by which an organism can be modified to include new genes designed with desired characteristics, is now routine practice in basic research laboratories. It has provided the means to produce large amounts of highly purified normal and mutant proteins for detailed analysis of their function in the organism.

Recent advances in this technology have also changed the course of medical research. Exciting new approaches are being developed to exploit the enormous potential of recombinant DNA research in the analysis of genetic disorders. The new ability to manipulate human genetic material has opened radically new avenues for diagnosis and treatment, and has far-reaching consequences for the future of medicine. Yet the basic principles of recombinant DNA, like the structure of DNA itself, are surprisingly simple.

Cloning DNA

Molecular cloning provides a means to exploit the rapid growth of bacterial cells for producing large amounts of identical DNA fragments, which alone have no capacity to reproduce themselves. The fragment of DNA to be amplified is first inserted into a cloning vector. The most popular vectors currently in use consist of either small circular DNA molecules (plasmids) or bacterial viruses (phage). The vectors contain genetic information that allows bacterial DNA replication machinery to copy them. After insertion of the foreign DNA, the plasmid or phage vector is re-introduced into a bacterial cell. The growing bacterial culture replicates the foreign DNA, along with the vector, in hundreds of copies per cell. This process yields multiple, identical clones of the original recombinant molecule. It is easy to harvest vectors from the bacterial culture, and release the amplified foreign DNA fragments with the same restriction enzyme used to insert the original DNA fragment into the vector (Figure 4, top). The power of molecular cloning is remarkable: a liter of bacterial cells engineered to amplify a single fragment of clones human DNA can produce about ten times the amount of a specific DNA segment than could be purified from the total cellular content of the entire human body.

For analysis of long stretches of DNA, eukaryotic vectors that can grow in yeast have been developed which can hold megabases of foreign DNA. These vectors mimic yeast chromosomal structure, so that they are replicated along with the native yeast chromosomes every time a yeast cell divides. Yeast Artifical Chromosomes, or YACs, are often the only way to clone extremely large genes including huge introns all in one continuous piece. YACs also provide a way to propagate DNA in a eukaryotic cell, where DNA modification, an important part of the eukaryotic genetic regulatory machinery, is more likely to be retained (more on this later). YACs are increasingly useful in the many Genome Projects underway, as we aim to understand the metastructure of chromosomes, where the placement and arrangement of genes within the "junk" DNA surrounding them may hold as yet undiscovered regulatory information for packaging and accessibility.

Amplification of Recombinant DNA

The DNA segment to be amplified is separated from surrounding genomic DNA by restriction enzyme cleavage, which often produces staggered or sticky ends. In the example illustrated here, the restriction enzyme EcoRI recognizes the palindromic sequence GAATTC, and cuts on each strand between G and A (the two strands of the genomic DNA are green and purple). The plasmid vector (brown) is prepared to accept the isolated genomic DNA fragment by cutting the circular plasmid DNA at a single site with the same restriction enzyme, generating sticky ends which are complementary to the sticky ends of the genomic DNA fragment. The cut genomic DNA and the linearized plasmid are mixed together in the presence of a ligase enzyme, which rejoins the bonds in the DNA backbone on each side of the plasmid-genomic DNA junction. This recombinant DNA molecule is then introduced into bacteria which are able to take up plasmid DNA, and then replicate the plasmid as the culture grows.

How was able to make a recombinant DNA?

Making Recombinant DNA

How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:

http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mga/ch10e1.gif


Inasmuch as the donor DNA was cut into many different fragments, most colonies will carry a different recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that the cloning method works because individual recombinant DNA molecules enter individual bacterial host cells, and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated like chemical reagents. Figure 10-1 on the following page gives a general outline of the approach.

The term recombinant DNA must be distinguished from the natural DNA recombinants that result from crossing-over between homologous chromosomes in both eukaryotes and prokaryotes. Recombinant DNA in the sense being used in this chapter is an unnatural union of DNAs from nonhomologous sources, usually from different organisms. Some geneticists prefer the alternative name chimeric DNA, after the mythological Greek monster Chimera. Down through the ages, the Chimera has stood as the symbol of an impossible biological union, a combination of parts of different animals. Likewise, recombinant DNA is a DNA chimera and would be impossible without the experimental manipulation that we call recombinant DNA technology.
Isolating DNA

The first step in making recombinant DNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types are generally the ones required for analysis. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 10-2 on page 303. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.top link
Cutting DNA

The breakthrough that made recombinant DNA technology possible was the discovery and characterization of restriction enzymes. Restriction enzymes are produced by bacteria as a defense mechanism against phages. The enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Importantly, restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation. Any DNA molecule, from viruses to humans, contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of size suitable for cloning. Restriction sites are not relevant to the function of the organism, nor would they be cut in vivo, because most organisms do not have restriction enzymes.

Let's look at an example: the restriction enzyme EcoRI (from E. coli) recognizes the following sixnucleotide-pair sequence in the DNA of any organism:

more detail : http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mga.section.1549

Is There a 'Bad Driver' Gene?


Is There a 'Bad Driver' Gene?
1 in 3 people have DNA that may make things tougher behind the wheel, researchers say

In a small study, researchers found that people with a gene variation performed 20 percent worse on simulated driving tests and did as poorly a few days later. Almost one in three Americans have the variation, the team said.

"These people make more errors from the get-go, and they forget more of what they learned after time away," said Dr. Steven Cramer, neurology associate professor at the University of California at Irvine and senior author of a study published recently in the journal Cerebral Cortex, in a statement.

The study authors say the gene variation lowers available levels of a protein that boosts memory by helping brain cells talk to one another and work properly.

Earlier research has suggested people with the variation engage smaller areas of the brain when they take on tasks.

"We wanted to study motor behavior, something more complex than finger-tapping," said Stephanie McHughen, a graduate student and lead author of the study in a statement. "Driving seemed like a good choice because it has a learning curve, and it's something most people know how to do."

Twenty-nine people took a driving test on a simulator, including seven with the gene variation. They had to learn to "drive" on a track that included tough-to-navigate curves and turns. They came back four days later to retake the test.

Those with the variant did worse and failed to remember as much the second time around as the others. "Behavior derives from dozens and dozens of neurophysiologic events, so it's somewhat surprising this exercise bore fruit," Cramer said.

But don't be alarmed if you think you have this gene variation -- it has it's good side. The researcher say the gene also slows mental decline for people with conditions such as Parkinson's disease, Huntington's disease or multiple sclerosis.

"It's as if nature is trying to determine the best approach," Cramer said. "If you want to learn a new skill or have had a stroke and need to regenerate brain cells, there's evidence that having the variant is not good. But if you've got a disease that affects cognitive function, there's evidence it can act in your favor. The variant brings a different balance between flexibility and stability."

FDA OKs New HPV Vaccine Cervarix


Cervarix targets two HPV strains, HPV 16 and HPV 18, which are leading causes of cervical cancer. Cervarix is approved to help prevent cervical precancers and cervical cancers associated with those two types of HPV.

HPV infection is common; the virus is sexually transmitted. Most women who get infected don't develop cervical cancer, and there are other causes of cervical cancer.

In clinical trials, Cervarix was shown to be 93% effective in preventing cervical precancers associated with HPV 16 or HPV18 in women with no evidence of current or previous infection with one of those two HPV types, according to GlaxoSmithKline, the drug company that makes Cervarix.

Cervarix is the second FDA-approved HPV vaccine. In June 2006, the FDA approved the first HPV vaccine, Gardasil, which targets four strains of HPV: HPV 6, HPV 11, HPV 16, and HPV 18.

BioAnalyst Researcher (m/f)

Job Description:

BioAnalysis Services team provides a customer-oriented working environment and dedicated services to BioScience R&D programs at competitive cost and time. The team actively participates to the definition of new approaches for the implementation of new technologies within research and frequently interact with bioinformatics competences and various researchers throughout the research departments of BioScience. In order to make competitive use of the complex in silico data coming from next-generation sequencing technologies, BioAnalysis Services will develop its competences in advanced areas such as systems biology and comparative genomics. Therefore we are looking for a BioAnalyst Researcher who will conduct the following tasks:
- Deliver genomic annotations for our crops of interest.
- Explore, test and evaluate existing tools to assemble NGS data
- Develop a quality control pipeline for this NGS data
- Develop tools/applications that will streamline our NGS platform
- Propose new strategies in order to extract information from the existing data
- Develop good relationships with the internal and external users/providers of NGS data
- Develop a good knowledge of the existing NGS platforms and keep up to date with this fast evolving area
Requirements:

You have got one of the following degrees:
- Master in bioinformatics
- Master in research fields related to biotechnology (molecular biology, genetics, genomics), but with a high interest in informatics
- Master in informatics/computer science or related research fields with a high interest in biotechnology/biology

Furtherone you have got a good knowledge of a programming language (Perl, JAVA, C#, etc) and databases. Knowledge of plant biology molecular genetics, marker technologies, protein biochemistry, genome engineering, plant breeding) and BioAnalysis (sequence analysis, genetic mapping, genome annotation, comparative genomics and systems biology).
Creative and flexible thinking, accuracy, autonomy and good communication skills for close relations with researchers and for the good understanding of the needs and a team player are also skills which you posses.

http://www.genomeweb.com/node/925295

Protein Chemist

Description: We have an opportunity for an experienced protein chemist to participate in advancing Seattle Genetics’ industry-leading Antibody Drug Conjugate Technology in the Chemistry Department. We seek an individual who will enjoy working at the bench in a collaborative fashion alongside scientists with a variety of backgrounds and specialties. This position will allow a scientist to contribute to new antibody-drug conjugate programs from early discovery steps through IND submission.

RESPONSIBILITIES:

* Preparing antibody-drug conjugates (ADCs) at a variety of scales using established and experimental methodologies.
* Characterizing ADCs by a variety of techniques, including high resolution mass spectrometry and various chromatographic methods.
* Purifying antibodies and other recombinantly expressed proteins.
* Working with other scientists and departments in support of ADC discovery projects.
* Independent research to advance our understanding of ADC performance and improve future ADC programs.

Required Skills:

REQUIREMENTS:

* Ph.D. in Biochemistry, Chemistry, or a related field, or a B.S. degree with at least 8 years experience in the biopharmaceutical industry.
* Experience with chemical modification of proteins.
* Experience in the biochemical and biophysical characterization of purified proteins, including HPLC methods, SDS-PAGE, and spectroscopic methods.
* Good communication and organizational skills and the ability to interface constructively with other scientists.
* Desire to learn new techniques and expand one's skill set.

Please click on the following link to apply:
http://ats.staffxl.com/ATS/PortalViewRequirement.do?reqGK=43019

European Co-Ordination Of Antimalarial Drug Discovery
The European Commission is funding a two year, €500,000 project to co-ordinate European and international research into the development of new drugs to treat malaria. The CRIMALDDI project (Coordination, Rationalisation and Integration of Antimalarial Drug Discovery and Development Initiatives) is being led by the Liverpool School of Tropical Medicine (LSTM) and brings together key players in ....

PPAR Inhibiors in Cancer Treatment

Poly (ADP-Ribose) Polymerase (PARP) is a protein cells use to repair genetic injuries naturally. But cancer cells also use this protein to repair their own DNA damage. Inhibiting this action allows chemotherapy and radiation to do its job against cancers resulting from genetic mutation.

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!