Structural Biochemistry/DNA recombinant techniques

=Overview= In many areas of biochemical research, retrieving enough quantities of a substance under study is a challenge. For instance, a maximum of 7 mg of DNA polymerase I can be obtained from a 10 L culture of E. coli grown with the highest dilution of ~10^10 cells * mL^-1, and still, other proteins present yield lower amounts. DNA recombinant techniques, also known as genetic engineering and molecular cloning, allow scientists to manipulate, duplicate and apply DNA for research. Deletions, insertions and substitutions are the most useful changes implemented for the synthesis of new genes. Such techniques usually include the cloning or amplification of sequences for further study. Restriction enzymes and DNA ligase play a  vital  role in the production of recombinant DNA. Polymerase chain reaction (PCR) is often used on interested sequences to be cut by nucleases and then cloned by phages, BACs or YACs. Using a DNA or RNA probe, specific genes can be cloned from a genomic library. The fundamental idea of DNA recombinant techniques is to input a DNA segment into a replicating DNA molecule, also known as a cloning vector or vehicle, so a DNA segment is replicated with the vector.The usage of various recombinant techniques can lead scientists to new understandings of how DNA affects us and other organisms. This section describes the various techniques involved in genetic engineering, and its application to many experiments carried out in biochemistry.

Cloning Vectors
A vector is a DNA molecule that can be used to insert a DNA sequence into a cell. Vectors are used for replication. Usual vectors in laboratories, are plasmids, viruses (Lambda Phage),and artificial chromosomes. Vectors must be capable of being replicated by the cell.

Plasmid-Based Cloning Vectors
Plasmids are doubled stranded, and commonly circular. They are predisposed with genetic material, like the replication origin (where DNA replication starts) to independently replicate inside of a bacterial host or yeast. Although plasmids may act as parasites, they are beneficial in ways like antibiotic resistance.

On one hand, some plasmids are present in quantities of one or two in a cell and replicate once per cell division and are known to be under stringent control. On the other hand, most plasmids utilized in genetic engineering are under relaxed control where as little as 10 and as many as 700 copies of plasmids can be present in a cell. In addition, when protein synthesis comes to a halt due to inhibition by an antibiotic, cell division also stops and these plasmids can replicate upwards of 3,000 copies in a cell. The types of plasmids synthesized from genetic engineering are under relaxed control, and contain genes that are resistant to antibiotics, while carrying endonuclease sites. These endonuclease sites facilitate the insertion of the DNA desired to be copied. A multiple cloning site, or a polylinker is a small section of a DNA that has an array of restriction sites not found anywhere else in the plasmid. pUC18 ("plasmid Universal Cloning") is a commonly used vector from E. coli. The following link provides more information and a schematic of pUC18.

In 1973, Herbert Boyer and Stanley Cohen demonstrated the first genetically mixed plasmid in DNA cloning. When a host bacterium is mixed with a plasmid, the conditions for optimum conditions is when divalent cations like calcium and heating to ~42 degrees Celsius is applied. This condition allows cell membranes to become more permeable to DNA and are called transformation competent. Once a plasmid vector is absorbed, it is permanently established in the bacterial host with only ~0.1% efficiency. Plasmid vectors are incapable of being used to duplicate DNA more than a certain size, because the time it takes for plasmid replication is directly proportional to the plasmid size.

Virus-Based Cloning Vectors
Another method to clone DNA's that are larger in size is using a cloning vehicle named bacteriophage lambda. The central third of the virus's genome space can be inserted by DNA's of larger sizes because that area is not necessary during phage infection. In vitro methods can help the insertion of the chimeric phage DNA by infecting the host cells through phages. Utilization of phages as cloning vectors has advantages in that the chimeric DNA can be made in large amounts and easily purified. In addition, scientists can make use of lambda phages for longer DNA inserts. The only requirement that is needed to allow the viral tool to insert DNA into the heads of phage is the 16-bp sequence known as the cos site at each end. These ends need to be at least 36-51 kb apart, and a combination of two cos sites in vitro creates a cosmid vector. Cosmids do not have phage genes and therefore can make plasmids. The following link is a cartoon picture of a bacteriophage lambda and its important features labeled.

M13- filamentous bacteriophage is a another cloning vector that is single-stranded and circular DNA in a protein tube. The amount of identical helical protein subunits are dependent on the length of phage DNA being coated, but is is normally ~2700 subunits. A longer unit of phage molecule results when an insertion of foreign DNA in a nonessential region occurs. The phages directly make single-stranded DNA that a certain technique requires, even though M13 cannot maintain DNA inserts larger than 1kb. This demonstrates a visualization of what an M13 looks like.

Another method of virus based cloning is baculoviruses, which are an array of large and pathogenic viruses that infect insects but not vertebrates, resulting in an easy way to culture them in lab. Like other viruses, a segment of the double stranded DNA that forms the genome of the viruses is not important for viral replication and can be replaced by foreign DNA upward to 15kb. This is an electron micrograph of the baculovirus.

YAC and BAC Vectors
In order to accommodate DNA segments larger than those carried by cosmids, yeast artificial chromosomes (YACs) and in bacterial artificial chromosomes (BACs). YACs contain all molecular necessities of required for replication for yeast such as the replication origin, autonomously replicating sequence (ARS), a centromere, and [|telomeres]. BACs replicate in E. Coli, and are found from plasmids that normally replicate long segments of DNA. BAC vectors have the bare minimum sequences necessary for self replication, copy number control, and regulated plasmid separation during cell division. The following link is a YAC diagram and [|BAC diagram].

Gene Manipulation
Through restriction endonucleases, a sequence-defined fragment can be retrieved when cloning a DNA. Oftentimes, reestriction endonucleases split a double stranded region of DNA at specific sites to result in single-stranded ends that complement one another. In 1972, Janet Mertz and Ron Davis shown that a restriction fragment can be inputted into a split made in a cloning vector by the same restriction enzyme. Complementary endings of two DNAs are covalently spliced with the help of DNA ligase. DNA insert can be extracted from a cloned vector by splitting it with the same restriction enzyme by making a chimeric vector.

Terminal deoxynucleotidyl transferase (TdT), was a technique produced by Dale Kaiser and Paul Berg who determined that if foreign DNA and cloning vector do not have a common restriction site, they can be spliced and this procedure can be carried out. The enzyme adds nucleotides to the 3'-terminal OH group of a DNA chain, and it is the only known DNA polymerase where a template is not required. In addition, the cloning vector is cleaved through an enzyme at a specific site, and the 3' ends are extended with poly(dA) tails. Finally, the homopolymer tails are heated and then cooled down, and any gaps due to differences with the other strand is filled in by DNA polymerase I and DNA ligase joins the two together.

Compared to other techniques gene manipulation takes out the restriction sites that were used to generate the foreign DNA insertion. Therefore, it can be hard to retrieve the insert from the cloned vector. However, this problem can be prevented by which a chemically synthesized linker matches the restriction site of the cloning vector is joined with the two ends of the foreign DNA. The adhesion is through the use of T4 DNA ligase and then split with the restriction enzyme. The schematic of splicing DNA with terminal transferase can be seen.

Cells Transformed Need to be Selected
It is difficult in choosing the host that have been transformed by the vector due to the low efficiency in transformation and constriction of chimeric vectors. As a result, in plasmid transformation, a double screen using antibiotics or color producing substrates is used. An example would be pUC18 plasmid that contains lacZ' since lacZ' gene codes for the enzyme beta-galactosidase. That gene initiates the hydrolysis of the bond from O1 of sugar beta-D-galactose to a substituent. Therefore, when grown in 5-bromo-4chloro-3-indolyl-beta-D-galactoside short handed for X-gal and hydrolysis, the colorless substance turns blue. Two different scenarios can occur: When E. coli is transformed by an unmodified pUC18 plasmid form blue colonies, then when E. coli is transformed by a pUC18 plasmid containing a foreign DNA insert in a polylinker, it is colorless due to the insert conflicting with the encoding sequence of lacZ' gene. Addition of an ampicillin allows the exclusion of bacteria that would normally become colorless when X-gal is added. This is due to the fact that bacteria that will have color have resistance intact from amp^R gene. These amp^R genes are known as selectable markers. Some of the genetically engineered lambda phages have restriction sites that border the available central third of a phage genome, and that segment can be replaced through a DNA insert. Lambda phages that are unable to obtain a foreign DNA are too short and therefore are unable to become an infectious phage. Cosmids in particular can support conception of larger DNA inserts due to their loss of DNA during random deletion and are not recovered.

Plasmid Vectors
Plasmids are naturally occurring circular pieces of DNA that are found in bacteria. Plasmids can be used as vectors to incorporate and replicate a DNA insert of interest by joining that DNA insert into the plasmid DNA. The vector is prepared to accept a DNA insert by treatment with a restriction enzyme (like EcoRI), which cleaves it at specific sites and leaves complementary single stranded "sticky ends." The DNA fragment to be inserted is treated with the same restriction enzyme so that it has complementary ends to those of the vector. DNA ligase is then used to join the DNA insert into the vector plasmid, resulting in recombination. The recombinant plasmid DNA can be cloned and amplified as the bacteria host colony grows.



Enzymes used in Recombinant DNA Technology
1. Type II restriction endonucleases always recognize palindromic sequences and the are able to cleave the DNA strand. In other words, they break the phosphodiester bonds of specific base sequences in the DNA.

2. DNA ligase is the opposite of Type II restriction endonuclease in that it joins two DNA molecules or fragments together.

3. DNA Polymerase is an enzyme that is able to use a template DNA and synthesize a complimentary strand by adding nucleotides to the 3' ends.

4. Reverse transcriptase is an enzyme that creates a DNA from an RNA molecule. Reverse transcriptase does the opposite of RNA polymerase, which takes DNA and makes RNA.

5. Polynucleotide kinase adds a phosphate to the 5' -OH end of a polynucleotide. it is useful in radioactively label DNA or permit ligation. 6. Voet, Donald, Judith G. Voet. Biochemistry 3rd ed. New Jersey: John Wiley & Sons, Inc, 2004. Print.