Genetic Engineering well explained

Genetic engineering allows the insertion of a foreign gene into cells and this makes them capable of producing a new and different protein.

Cloning of a Gene

Formation of recombinant DNA

Recombinant DNA (rDNA) or chimaeric contains genes from two or more different sources. To make rDNA, first a vector is selected. By means of vector rDNA is introduced into the host cell. One common type of vector is a plasmid. Plasmids are small accessory rings of DNA, which is not part of bacterial chromosome and can replicate independently. Making many identical copies of a molecule is called cloning. Phages are viruses which can inject their DNA into bacteria for replication. The piece of DNA, (foreign gene) of phage combines with plasmid. The modified plasmid is called rDNA. When bacteria divide, the plasmids are replicated. Eventually there are many copies of the plasmid and therefore many copies of the foreign gene. The gene is now said to have been cloned.

Genetic engineering in bacteria can be divided into five stages. The order of stages are:

(a) Obtain a copy of the required gene from the DNA of the donor organism.

(b) Place the gene in a vector i.e. plasmid.

(c) Use the plasmid to introduce the gene into the host cell (bacterium).

(d) Select the bacteria which have taken up the DNA i.e. foreign DNA.

(e) Clone the gene.

Use of Restriction Endonucleases and Plasmids in rDNA Technology

A specific piece of DNA containing the gene of interest must be cut out of a chromosome and “pasted” into a bacterial plasmid. The cutting tools of recombinant technology are bacterial enzymes. These are restriction endonucleases or restriction enzymes. In 1970 Hamilton O. Smith, at John Hopkins University, isolated the restriction enzymes. Restriction enzymes occur naturally in bacteria, where they stop viral reproduction by cutting up viral DNA. They are called restriction enzymes because they restrict the growth of viruses. Bacteria produce a variety of restriction enzymes. These cut the DNA at very specific sites characterized by specific sequence of four or six nucleotide arranged symmetrically in the reverse order. Such sequence are known as palindromic sequence or recognition sequence. For example EcoRI, is a commonly used restriction enzyme, when double stranded DNA has AATT sequence of bases at cleavage site.

There are several hundred restriction enzymes and about a hundred different recognition sequences known. In the figure here, step (1) shows a piece of DNA containing two copies of a recognition sequence. In this case, the restriction enzyme will cut the DNA strands in the four places within each recognition sequence where the bases A and G lie next to each other. (The places where DNA is cut are called restriction sites.) (2) The result is a set of double-stranded DNA fragments with single-stranded ends, called “sticky ends”. Sticky ends are the key to joining DNA restriction fragments originating from different sources, even from different species. These short extensions will form hydrogen-bonded base pairs with complementary single stranded stretches of DNA.

Step (3) shows a piece of DNA (pink) from another source. Notice that the pink DNA has single-stranded ends identical in base sequence to the sticky ends on the blue DNA. The pink, “foreign” DNA has ends with this particular base sequence because it was cut from a larger molecule with the same restriction enzyme .used to cut the blue DNA. (4) The complementary ends on the blue and pink fragments allow them to stick together by base pairing. (The hydrogen bonds that hold the base pairs together are not shown.)

Steps showing formation of rDNA using restriction enzyme and DNA ligase

The union between the blue and pink DNA fragments shown in step 4 (as you can see in the figure) is only temporary, because only a few hydrogen bonds hold the fragments together. The union can be made permanent, however, by the "pasting" enzyme DNA ligase. This enzyme, which the cell normally uses in DNA replication catalyzes the formation of covalent bonds between adjacent nucleotides, sealing the breaks in the DNA strands. (5) The final outcome is recombinant DNA, a DNA molecule carrying a new combination of genes.

Genes Can Be Cloned In Recombinant Plasmids

This figure here illustrates a way to make many copies of the gene using the techniques we have been discussing.

Cloning a Gene in a Bacterial Plasmid

In step (1), the biologist isolates two kinds of DNA: the bacterial plasmid that will serve as the vector, and eukaryotic DNA containing the gene. In step (2), the researcher treats both the plasmid and the human DNA with the restriction enzyme. The enzyme cuts the plasmid DNA at one specific restriction site. It also cuts the human DNA, generating many thousands of fragments; one of these fragments carries the insulin gene. In making the cuts, the restriction enzyme creates sticky end on both the human DNA fragments and the plasmid. For simplicity, the figure here shows the step-by-step processing of one human DNA fragment and one plasmid, but actually millions of plasmids and human DNA fragments are treated simultaneously. In step (3) the human DNA is mixed with the cut plasmid. The sticky ends of the plasmid base pair with the complementary sticky ends of the human DNA fragment. In step (4), the enzyme DNA ligase joins the two DNA molecules by covalent bonds, and the result is a recombinant DNA plasmid containing insulin gene. In step (5), the recombinant plasmid is added to a bacterium. Under the right conditions, the bacterium will take up the plasmid DNA from solution by the process of transformation. The last step shown here, step (6), is the actual gene cloning, the production of multiple copies of the gene. The bacterium, with its recombinant plasmid, is allowed to reproduce. As the bacterium forms a cell clone, any genes carried by the recombinant plasmid are also cloned.

Expression of Recombinant DNA

Plasmid library: Bacterial cells take up recombined plasmids, especially if they are treated with calcium chloride to make them more permeable. Thereafter, as the host cell reproduces, a bacterial clone forms. Each of the bacteria contains the foreign gene, which is behaving just as if it were in original cell. The investigator can recover either the cloned gene, or a protein product from this bacterial clone.

Preparation of a genomic library

Plasmids can be used as vectors for carrying foreign DNA, and so can viruses, such as the bacteriophage known as lambda. After lambda attaches to a cell, the DNA is released from the virus and enters the bacterium. Here it may direct the reproduction of many more viruses. Each virus in the bacteriophage clone contains a copy of the foreign gene. Notice that a clone is a large number of molecules (i.e. cloned genes) or viruses (i.e. cloned bacteriophages) or cells (i.e. cloned bacteria) that are identical to an original specimen.

Using a Genomic Library

Phase library: A genome is the full set of genes of an individual. A genomic library is a collection of bacterial or bacteriophage clones; each clone contains a particular segment of the DNA from a foreign cell. When you make a genomic library, an organism's DNA is simply sliced up into pieces, and the pieces are put into vectors (i.e. plasmids or viruses) that are taken up by the host bacteria. The entire collection of bacterial or bacteriophage clones that result therefore contains all the genes of that organism.

Reverse Transcriptase helps make DNA for cloning

In order for mammalian gene expression to occur in a bacterium, the gene has to be accompanied by the proper regulatory regions. Also, the gene should not contain introns (non-coding region) because bacterial cells do not have the necessary enzymes to process primary mRNA. It is possible to make a mammalian genome that lacks introns. The enzyme called ‘reverse transcriptase’ can be used to make a DNA copy of all the mature mRNA, molecules from a cell. This DNA molecule, called complementary DNA (cDNA), does not contain introns.

Use a Particular Probe to Search a Genomic Library for a Certain Gene

A probe is a single-stranded nucleotide sequence of RNA that will hybridize (pair) with a certain piece of DNA. Methods for detecting genes all depend on base paring between the gene and a complementary sequence on another nucleic acid molecule, either DNA or RNA. When at least part of the nucleotide sequence of a gene is already known or can be guessed, this information can be used to advantage. Taking a simplified example, if we know at our hypothetical gene V Contains the sequence TAGGCT, a biochemist can use nucleotide labeled with a radio Active isotope to synthesize a short RNA molecule with a complementary sequence (AUCCGA) (as shown in the figure). This short labelled nucleic acid molecule is called probe because it is used to find a specific gene (or other nucleotide sequence) within a mass of DNA. In practice probe molecule would be considerably longer than six nucleotide. Location of the probe is possible because me probe is either radioactive or fluorescent. As illustrated in 2nd figure bacterial cells, each carrying a particular DNA fragment, can be plated onto agar in a Petri dish. After the probe hybridizes with the gene of interest, the gene can be isolated from the fragment. Now this particular fragment cab be cloned further or even analyzed for its particular DNA sequence. 

Identification of a cloned gene

Replicating Small DNA Segments

The polymerase chain reaction (PCR) was developed by Kary B. Mullis in 1983. It can create millions of copies of a single gene or any specific piece of DNA in laboratory glassware e.g. a test tube. PCR is very specific. The targeted DNA sequence can be less than one part in a million of the total DNA sample. This means that a single gene among all the human genes can be amplified (copied) using PCR.

Polymerase chain reaction (PCR)

PCR takes its name from DNA polymerase, the enzyme that carries out DNA replication in a cell. It is considered a chain reaction because DNA polymerase will carry out replication over and over again, until there are millions of copies of the targeted DNA. PCR does not replace gene cloning; cloning is still used wherever a large quantity of a protein product is needed, or if the DNA needs to be in its accustomed configuration before being sequenced.

Before carrying out PCR, primers sequences of about 20 bases that are complementary to the bases on either side of the “target DNA” must be available. The primers are needed because DNA polymerase does not start the replication process; it only continues or extends the process. After the primers bind by the complimentary base pairing to the DNA strand, DNA polymerase copies the target DNA, as shown in the figure.

PCR has been in use for several years, and now almost every laboratory has automated PCR machines or thermocycler to carry out the procedure. Automation became possible after a temperature-insensitive (thermostable) DNA polymerase also known as Taq polymerase was extracted from the bacterium Thermus aquaticus, which lives in hot springs. This enzyme can withstand the high temperature used to separate the double-stranded DNA; therefore, replication need not to be interrupted by the need to add more enzyme.

Analyzing DNA

DNA can be subjected to DNA fingerprinting, a process described in figure. When the DNA of an organism is treated with restriction enzymes, the result is a unique collection of different-sized fragments called restriction fragments, which reflect specific sequence of nucleotide. Therefore, restriction fragment length polymorphisms or RFLPs, (pronounced “rif-lips") exist between individuals. During a process called gel electrophoresis, the fragments can be separated according to their lengths, and the result is a No. of bands that are so close together that they appear as a smear. However, the use of probes for genetic markers results in a distinctive pattern that can be recorded on X-ray film.

PCR amplification and analysis has been used (1) to diagnose viral infections, genetic disorders, and cancer; (2) in forensic laboratories to identify criminals; and (3) to determine the evolutionary relationships of various organisms. When the amplified DNA matches that of a virus, mutated gene or oncogene (cancer gene), then we know that a viral infection, genetic disorder, or cancer is present. To determine evolutionary relationships, it is often necessary to sequence the DNA. Sequencing mitochondrial DNA segments helped to determine the evolutionary history of human populations. It has even made possible to sequence DNA taken from a 17 to 20 billion year old plant fossil following PCR amplification. Nucleotide sequences of DNA sequencer make use of computers.

DNA Sequencing Technique Gel-electrophoresis

This laboratory procedure uses an electric field to move molecules through a viscous gel and separate them according to their size, shape and net surface charge. Often the gel is sandwiched between glass or plastic plates to form a viscous slab. The two ends of the slab are suspended in two salt solutions that are connected by electrodes to a power source. When voltage is applied to the apparatus, the molecules present in the gel migrate through the electric field according to their individual charge, and they move away from one another in the gel. Later on, the molecules can be pinpointed by staining the gel after a predetermined period of electrophoresis.

Gel electrophoresis