Recombinant DNA and Gene Cloning

Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule.

Making Recombinant DNA (rDNA): An Overview

• Treat DNA from both sources with the same restriction endonuclease (BamHI in this case).
• BamHI cuts the same site on both molecules

  5' GGATCC 3'
  3' CCTAGG 5'

• The ends of the cut have an overhanging piece of single-stranded DNA.
• These are called "sticky ends" because they are able to base pair with any DNA molecule containing the complementary sticky end.
• In this case, both DNA preparations have complementary sticky ends and thus can pair with each other when mixed.
• a DNA ligase covalently links the two into a molecule of recombinant DNA.

To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. Producing many identical copies of the same recombinant molecule is called cloning. Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done in vivo.
Cloning in vivo can be done in

• unicellular microbes like E. coli
• unicellular eukaryotes like yeast and
• in mammalian cells grown in tissue culture.

In every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But here let us examine an example of cloning using E. coli as the host and a plasmid as the vector.

Plasmids

Electron micrograph of an E. coli cell ruptured to release its DNA. The tangle is a portion of a single DNA molecule containing over 4.6 million base pairs encoding approximately 4,300 genes. The small circlets are plasmids. (Courtesy of Huntington Potter and David Dressler, Harvard Medical School.)

Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome.
They:

• are small (a few thousand base pairs)
• usually carry only one or a few genes
• are circular
• have a single origin of replication

Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.
Genes on plasmids with high numbers of copies are usually expressed at high levels. In nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from one or more antibiotics.
Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes.

An Example

(courtesy of David Miklos and Greg Freyer of the Cold Spring Harbor Laboratory, who used these plasmids as the basis of a laboratory introduction to recombinant DNA technology that every serious biology student — high school or college — should experience!)

pAMP

• 4539 base pairs
• a single replication origin
• a gene (amp^r)conferring resistance to the antibiotic ampicillin (a relative of penicillin)
• a single occurrence of the sequence

  5' GGATCC 3'
  3' CCTAGG 5'

  that, as we saw above, is cut by the restriction enzyme BamHI
• a single occurrence of the sequence

  5' AAGCTT 3'
  3' TTCGAA 5'

  that is cut by the restriction enzyme HindIII

Treatment of pAMP with a mixture of BamHI and HindIII produces:

• a fragment of 3755 base pairs carrying both the amp^r gene and the replication origin
• a fragment of 784 base pairs
• both fragments have sticky ends

pKAN

• 4207 base pairs
• a single replication origin
• a gene (kan^r) conferring resistance to the antibiotic kanamycin.
• a single site cut by BamHI
• a single site cut by HindIII

Treatment of pKAN with a mixture of BamHI and HindIII produces:

• a fragment of 2332 base pairs
• a fragment of 1875 base pairs with the kan^r gene (but no origin of replication)
• both fragments have sticky ends

These fragments can be visualized by subjecting the digestion mixtures to electrophoresis in an agarose gel. Because of its negatively-charged phosphate groups, DNA migrates toward the positive electrode (anode) when a direct current is applied. The smaller the fragment, the farther it migrates in the gel.

Ligation Possibilities

If you remove the two restriction enzymes and provide the conditions for DNA ligase to do its work, the pieces of these plasmids can rejoin (thanks to the complementarity of their sticky ends).
Mixing the pKAN and pAMP fragments provides several (at least 10) possibilities of rejoined molecules. Some of these will not produce functional plasmids (molecules with two or with no replication origin cannot function).
One interesting possibility is the joining of

• the 3755-bp pAMP fragment (with amp^r and a replication origin) with the
• 1875-bp pKAN fragment (with kan^r)

Sealed with DNA ligase, these molecules are functioning plasmids that are capable of conferring resistance to both ampicillin and kanamycin. They are molecules of recombinant DNA.
Because the replication origin, which enables the molecule to function as a plasmid, was contributed by pAMP, pAMP is called the vector.

Transforming E. coli

Treatment of E. coli with the mixture of religated molecules will produce some colonies that are able to grow in the presence of both ampicillin and kanamycin.

• A suspension of E. coli is treated with the mixture of religated DNA molecules.
• The suspension is spread on the surface of agar containing both ampicillin and kanamycin.
• The next day, a few cells — resistant to both antibiotics — will have grown into visible colonies containing billions of transformed cells.
• Each colony represents a clone of transformed cells.

However, E. coli can be simultaneously transformed by more than one plasmid, so we must demonstrate that the transformed cells have acquired the recombinant plasmid.
Electrophoresis of the DNA from doubly-resistant colonies (clones) tells the story.

• Plasmid DNA from cells that acquired their resistance from a recombinant plasmid only show only the 3755-bp and 1875-bp bands (Clone 1, lane 3).
• Clone 2 (Lane 4) was simultaneous transformed by religated pAMP and pKAN. (We cannot tell if it took up the recombinant molecule as well.)
• Clone 3 (Lane 5) was transformed by the recombinant molecule as well as by an intact pKAN.

Cloning other Genes

The recombinant vector described above could itself be a useful tool for cloning other genes. Let us assume that within its kanamycin resistance gene (kan^r) there is a single occurrence of the sequence

5' GAATTC 3'
3' CTTAAG 5'

This is cut by the restriction enzyme EcoRI, producing sticky ends. If we treat any other sample of DNA, e.g., from human cells, with EcoRI, fragments with the same sticky ends will be formed. Mixed with EcoRI-treated plasmid and DNA ligase, a small number of the human molecules will become incorporated into the plasmid which can then be used to transform E. coli.
But how to detect those clones of E. coli that have been transformed by a plasmid carrying a piece of human DNA?
The key is that the EcoRI site is within the kan^r gene, so when a piece of human DNA is inserted there, the gene's function is destroyed.
All E. coli cells transformed by the vector, whether it carries human DNA or not, can grow in the presence of ampicillin. But E. coli cells transformed by a plasmid carrying human DNA will be unable to grow in the presence of kanamycin.
So,

• Spread a suspension of treated E. coli on agar containing ampicillin only
• grow overnight
• with a sterile toothpick transfer a small amount of each colony to an identified spot on agar containing kanamycin
• (do the same with another ampicillin plate)
• Incubate overnight

All those clones that continue to grow on ampicillin but fail to grow on kanamycin (here, clones 2, 5, and 8) have been transformed with a piece of human DNA.

Some recombinant DNA products being used in human therapy

Using procedures like this, many human genes have been cloned in E. coli or in yeast. This has made it possible — for the first time — to produce unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with a human gene are being used to manufacture more than 100 products for human therapy. Some examples:

• insulin for diabetics
• factor VIII for males suffering from hemophilia A
• factor IX for hemophilia B
• human growth hormone (HGH)
• erythropoietin (EPO) for treating anemia
• several types of interferons
• several interleukins
• granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant
• granulocyte colony-stimulating factor (G-CSF) for stimulating neutrophil production (e.g., after chemotherapy) and for mobilizing hematopoietic stem cells from the bone marrow into the blood.
• tissue plasminogen activator (TPA) for dissolving blood clots
• adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID)
• parathyroid hormone
• many monoclonal antibodies
• hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
• C1 inhibitor (C1INH) used to treat hereditary angioedema

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html

2. Growth Rate

Bacteria typically grow much faster than more complex organisms. E. coli grows rapidly at a rate of one generation per twenty minutes under typical growth conditions. This allows for preparation of log-phase (mid-way to maximum density) cultures overnight and genetic experimental results in mere hours instead of several days, months or years. Faster growth also means better production rates when cultures are used in scaled up fermentation processes.

3. Safety

E. coli is naturally found in the intestinal tracts of humans and animals where it helps provide nutrients (vitamins K and B12) to its host. There are many different strains of E. coli that may produce toxins or cause varying levels of infection if injested or allowed to invade other parts of the body. Despite the bad reputation of one particularly toxic strain (O157:H7), E. coli are generally relatively inocuous if handled with reasonable hygiene.

4. Conjugation and the Genome Sequence

The E. coli genome was the first to be completely sequenced. Genetic mapping in E. coli was made possible by the discovery of conjugation. E. coli is the most highly studied microorganism and an advanced knowledge of its protein expression mechanisms makes it simpler to use for experiments where expression of foreign proteins and selection of recombinants is essential.
_{source:
Weaver, R. and Hedrick, P. 1989. Genetics. Wm. C. Brown Publishers, Dubuque, IA, USA.}

5. Ability to Host Foreign DNA

Most gene cloning techniques were developed using this bacterium and are still more successful or effective in E. coli than in other microorganisms. E. coli is readily transformed with plasmids and other vectors, easily undergoes transduction, and preparation of competent cells (cells that will take up foreign DNA) is not complicated. Transformations with other microorganisms are often less successful.
http://biotech.about.com/od/technicaltheory/tp/Ecoli.htm

The microorganism Escherichia coli has a long history of use in the biotechnology industry and is still the microorganism of choice for most gene cloning experiments. Although E. coli is known to the general population for the infectious nature of one particular strain (0157:H7) few people are aware of how versatile and useful E. coli is to genetic research. There are several reasons E. coli became so widely used and is still a common host for recombinant DNA.

1. Genetic Simplicity

Bacteria make useful tools for genetic research because of their relatively small genome size compared to eukaryotes. E. coli cells only have about 4,400 genes whereas the human genome project has determined that humans contain approximately 30,000 genes. Also, bacteria, including E. coli, live their entire lifetime in a haploid state, with no second allele to mask the effects of mutations during protein engineering experiments.
_{source:
Weaver, R. and Hedrick, P. 1989. Genetics. Wm. C. Brown Publishers, Dubuque, IA, USA.
Madigan, M., Martinko, J. and Parker, J. 2000. Brock Biology of Micro-organisms, 9th ed. Prentice Hall, Upper Saddle River, NJ, USA.}

fact bioinformatics is a field having applications of Informatics (Information Technology) in biological sciences. It is becoming the most important and popular field after the starting of world’s heaviest and most costly project ‘Human Genome Project’.

The vast organisms present in nature and each having a different Genome, Proteome and a different way of interaction with the environment was difficult to accommodate by only human brain. So the field of BioInformatics was chosen to accommodate all by utilizing IT services for making different tools and techniques and also to develop databases that can have either information about one particular organism, species or system. Now a day, biological databases are the much heavier and by time they will grow much more according to the diversity of nature.

For all these purposes many agencies, either they are Pharmacist, Chemist, textile or any agency making use of natural treasures for human benefits, are starting projects and most rely on funding universities to complete their projects. New and advanced tools of BioInformatics are helping them to discover any problem in the genome of any organism to develop organism specific drugs, use of microorganisms for developing drug and immunity to changing environment and most importantly, how life started and how humans evolved.

Bioinformatics
Bioinformatics is the combination of biology and information technology. The
discipline encompasses any computational tools and methods used to manage,
analyze and manipulate large sets of biological data. Essentially, bioinformatics has
three components:

1. The creation of databases allowing the storage and management of large biological data sets.
   
2. The development of algorithms and statistics to determine relationships among members of large data sets.
3. The use of these tools for the analysis and interpretation of various types of biological data, including DNA, RNA and protein sequences, protein structures, gene expression profiles, and biochemical pathways.
   

The term bioinformatics first came into use in the 1990s and was originally synonymous with the management and analysis of DNA, RNA and protein sequence data. Computational tools for sequence analysis had been available since the 1960s, but this was a minority interest until advances in sequencing technology led to a rapid expansion in the number of stored sequences in databases such as GenBank. Now, the term has expanded to incorporate many other types of biological data, for example protein structures, gene expression profiles and protein interactions. Each of these areas requires its own set of databases, algorithms and statistical methods. Bioinformatics is largely, although not exclusively, a computer-based discipline.

Computers are important in bioinformatics for two reasons:

First, many bioinformatics problems require the same task to be repeated millions of times. For example, comparing a new sequence to every other sequence stored in a database or comparing a group of sequences systematically to determine evolutionary relationships. In such cases, the ability of computers to process information and test alternative solutions rapidly is indispensable.
Second, computers are required for their problem-solving power.
Typical problems that might be addressed using bioinformatics could include solving the folding
pathways of protein given its amino acid sequence, or deducing a biochemical pathway given a collection of RNA expression profiles.

Computers can help with such problems, but it is important to note that expert input and
robust original data are also required.
The future of bioinformatics is integration. For example, integration of a wide variety of data sources such as clinical and genomic data will allow us to use disease symptoms to predict genetic mutations and vice versa. The integration of GIS data,such as maps, weather systems, with crop health and genotype data, will allow us to
predict successful outcomes of agriculture experiments.

Another future area of research in bioinformatics is large-scale comparative genomics. For example, the development of tools that can do 10-way comparisons of genomes will push forward the discovery rate in this field of bioinformatics. Along these lines, the modeling and visualization of full networks of complex systems could be used in the future to predict how the system (or cell) reacts to a drug for example.
A technical set of challenges faces bioinformatics and is being addressed by faster computers, technological advances in disk storage space, and increased bandwidth.

Finally, a key research question for the future of bioinformatics will be how to computationally
compare complex biological observations, such as gene expression patterns and protein networks. Bioinformatics is about converting biological observations to a model that a computer will understand. This is a very challenging task since biology can be very complex. This problem of how to digitize phenotypic data such as behavior, electrocardiograms, and crop health into a computer readable form offers exciting challenges for future Bioinformaticians.
(Thanx to Bibliotheca Alexandrina for this material)

define:Bioinformatics - Google Search

define:Bioinformatics - Google Search: "The collection, organization and analysis of large amounts of biological data, using networks of computers and databases."

define:Bioinformatics - Google Search

define:Bioinformatics - Google Search: "The collection, organization and analysis of large amounts of biological data, using networks of computers and databases"