Top 4 Ways Carried Out for DNA Cloning | Genetics

The process of DNA cloning is carried out in four ways: 1. DNA Cloning Inside Host Cell 2. Chemically by Polymerase Chain Reaction (PCR) 3. Reproductive Cloning 4. Therapeutic Cloning.

Way # 1. DNA Cloning Inside Host Cell:

DNA cloning is the selective amplification of a specific DNA fragment or a sequence for producing large quantities of the DNA fragment or sequence for detailed analysis of its structure and function. The DNA segment to be cloned is first linked to a vector DNA which can carry the foreign DNA into a host cell such as E. coli.

The two types of vectors are commonly used to clone DNAs within bacterial cells, either plasmid or the genome of bacterial virus λ. In both cases, after insertion of foreign DNA, the recombinant plasmid or phage DNA is allowed to infect a culture of bacterial cells.

When the foreign DNA segment is inside the bacterium, it is replicated along with the plasmid or the viral DNA and passed on to the daughter cells (Fig. 23.8). By this procedure, a single recombinant plasmid or viral genome present in one bacterial cell can be amplified to produce millions of copies of DNA.

Cloning DNA in Bacterial Plasmids:

A population of recombinant plasmids containing different fragments of foreign DNA from the genome of an organism are added to a culture of E. coli cells. Bacterial cells can take up DNA from the medium, undergoing transformation.

The bacterial cell membrane is normally not permeable to large molecules such as DNA fragments, but can be made permeable by a variety of methods such as exposure to salts, heat, or high voltage for becoming competent for transformation.

Presence of calcium ions in the medium and a brief heat shock induce uptake of DNA by bacteria. Usually only a small percentage of bacteria become competent for uptake of recombinant DNA molecules. Moreover, usually only a single DNA molecule will be taken up by a host bacterial cell undergoing transformation. Inside the E. coli cell, the recombinant plasmid replicates autonomously and is passed on to the progeny cells.

Thus, large quantities of the original DNA, that is clones, would be produced. Bacterial progeny is grown in the presence of the antibiotic, so that bacteria carrying the recombinant plasmid (with genes for antibiotic-resistance) are easily selected against bacteria without plasmid.

Many bacteria contain restriction systems which can influence the process of transformation through recognition and degradation of foreign DNA. Therefore, restriction-deficient strains of E. coli are preferred for use as transformable hosts.

The population of recombinant plasmids that was initially added to bacterial cultures contained different foreign DNA fragments from the organism’s genome. It is possible to search those very few bacterial cells that carry a particular recombinant plasmid with a specific sequence of interest. E. coli cells containing the different recombinant plasmids are grown on petri dishes at low density, so that the progeny of each bacterial cell, representing a clone of cells, is physically distant from the progeny (clones) of other cells.

Because a number of different recombinant plasmids were initially added to the culture medium, the resulting clones on the petri dish will have different foreign DNA fragments. After the various recombinant plasmids have produced separate colonies, it is now required to search the few colonies that contain the sequence of interest.

Screening of colonies for a particular DNA sequence is done by the combined techniques of replica plating and in situ hybridisation. A large number of petri dishes are prepared reducing the number of colonies on each petri dish, the colonies having the same position on each dish (Fig. 23.9). For localising the specific DNA sequence, the cells on one replica plate are lysed and DNA fixed on to the surface of a filter.

The DNA is denatured and subjected to in situ hybridisation. The filter is incubated with a labeled DNA probe whose sequence is complementary to the sequence of interest. After incubation, the un-hybridised probe is washed from the filter. The position of the labelled hybrid is determined by autoradiography.

The position of the clone carrying hybridised probe would correspond with identical clones on the original plate. Cells from these clones are then sub-cultured to produce large colonies. This process results in amplification of the recombinant DNA plasmid. The cells are harvested and recombinant DNA is separated.

The recombinant plasmids are then treated with the same restriction endonuclease that was initially used to insert foreign DNA. The cloned foreign DNA (sequence of interest) is then separated from the vector (plasmid) DNA by centrifugation.

Cloning DNA in Bacteriophage Genomes:

Unlike most other DNA viruses that have single-stranded DNA, bacteriophage λ has double- stranded linear DNA about 50 kb in length. Phage has a standard amount of DNA that can be ‘packaged’ inside the head. Some genes of bacteriophage are absolutely essential for replication of its genome, whereas others are dispensable. A bacteriophage cloning vector must contain the essential DNA sequences.

In phage λ, the central part of the genome is not required for replication or packaging of λ DNA molecules in E. coli, and can be discarded. A modified strain of phage λ is therefore, prepared for cloning experiments. It contains two cleavage sites for the enzyme EcoRI, which produces three large fragments in the genome. The two outer segments at either end of the linear λ genome contain all the information essential for its growth.

The central fragment which is dispensable is removed and replaced by DNA up to 25 kb in length. Recombinant DNA molecules can be packaged into phage heads in vitro. Phage particles with recombinant DNA are used to infect E. coli cells (Fig. 23.6). The foreign DNA is replicated along with viral DNA and produces a large number of progeny virus particles. The process accomplishes amplification of the foreign DNA segment.

Lysis of the bacterial cell which are used to infect new E. coli cells, releases the progeny viruses. A clear spot or plaque becomes visible in the petri dish at the site of infection. Each plaque contains numerous phage particles each carrying a single copy of the desired fragment. The fragment of interest is identified by replica plating and in situ hybridisation as described for cloning of recombinant plasmids.

Way # 2. Cloning by Polymerase Chain Reaction (PCR):

PCR allows DNA sequences to be selectively amplified millions of times in just a few hours without using bacterial host cells. PCR has had a profound impact upon molecular biology. The technique was developed by Kary Mullis in 1983 using a heat-stable DNA polymerase enzyme that is stable at temperatures higher than 90°C.

The basic PCR procedure involves separation of DNA strands at high temperature (denaturation), and use of synthetic sequences of single-stranded DNA to serve as primers (Fig. 23.10). A primer (a DNA or RNA primer) with an exposed 3′-OH end, is essentially required for DNA polymerase to start DNA synthesis.

Two oligonucleotide primers are used, 17-30 nucleotides in length, which flank the DNA target sequence that is to be amplified. One primer is complementary to one DNA strand at the beginning of the target region, the second primer is complementary to a sequence on the opposite DNA strand at the end of the target region.

The primers hybridise to opposite strands of the DNA after it has been denatured, then allowing DNA synthesis by the polymerase to proceed through the stretch between the two primers. The single-stranded DNA molecules are extended toward each other (extension), forming a double-stranded DNA molecule identical with each starting one.

Repeated cycles of heat denaturation, hybridisation with primers, and extension result in exponential accumulation of the PCR amplification product, that was the target DNA sequence. The temperature resistant DNA polymerase used to catalyse extension from DNA primers is called taq polymerase, isolated from the bacterium Thermus aquaticus growing in hot springs.

To start the PCR reaction, a small sample of target DNA is added to the test tube, mixed with all the four deoxyribonucleotides (building blocks of DNA), taq polymerase, two synthetic oligonucleotide primers that are complementary to DNA sequences at the 3′ ends of the region of the DNA to be amplified.

The first step consists of heating the mixture to 92 to 94°C to denature duplex DNA into single strands. In the second step the mixture is cooled to 50 to 65°C to allow primers to bind or anneal to the strands of the target DNA. In the third step polymerase adds nucleotides to the 3′ end of the primers at 72°C.

PCR is a very sensitive technique with many applications in molecular biology. Target sequences that are in extremely low copy number in a sample can be amplified, if the primers are designed specific for this sequence.

The important point to note is that, amplification is highly selective, only the DNA sequence located between the primers is amplified exponentially. Following amplification, the PCR products are loaded into the wells of an agarose gel for electrophoresis. The amplified products are visualised by staining with ethidium bromide.

Stringency in PCR Experiments:

Stringency refers to the specificity with which the target DNA sequence is detected by hybridisation to the nucleic acid probe. It is influenced by temperature and salt conditions prevailing in the hybridisation step, when primers anneal to target DNA strand, as also in the post hybridisation washes. At high stringency, only those sequences that are completely complementary will be bound.

At low stringency conditions, even partially matched sequences will show hybridisation. The design of oligonucleotide primers is critical to obtain maximum hybridisation specificity. Oligonucleotide primers hybridise more rapidly than primers with more nucleotides. The longer the oligonucleotide primers, the less chance there is that it will bind to sequences other than the target sequence under conditions of high stringency.

Way # 3. Reproductive Cloning:

The technique used to generate an animal that has genetic material identical to that in another animal, is called reproductive cloning. A well known example is that of the sheep named Dolly. She was created in 1996 by a procedure referred to as “somatic cell nuclear transfer” (SCNT).

The method involves transfer of genetic material from the nucleus of an adult donor cell, in Dolly’s case an udder cell, into an egg whose nucleus has been removed. The egg has no genetic material of its own. After transfer of donor nucleus from udder cell, the egg is stimulated to undergo cell division by treatment with chemicals or electric current.

The multicellular embryo thus consists of a clone of cells whose genetic material, the entire genome had originated from the donor nucleus. The offspring is thus identical to its single ‘parent’ that provided the nucleus. At the appropriate stage the cloned embryo is transferred to the uterus of a female host where it continues to grow and give birth to an offspring.

Traditionally the term clone has been used for descendents of a single cell that contain genetic material identical to that of the parent cell. It may be noted that the cloned embryo described above has also inherited mitochondrial DNA from the cytoplasm of the enucleated egg. Hence the resulting offspring is not a clone in the strict sense of the term, as it contains genetic material from two sources, the donor nucleus of an udder cell, as well as mitochondrial DNA from the egg.

The cloned sheep Dolly died after six years from cancer and arthritis. Her creation however, proved that genetic material from an adult specialised cell such as an udder cell could be reprogrammed to produce an entire new organism.

There was clear demonstration to dispel the earlier notion that in a specialised cell from heart, lung, udder or any type of cell, the genetic material was programmed permanently to express only the functions of its tissue of origin. A large number of animals including goats, sheep, dog, cows and cats have been cloned by the nucleus transfer technique.

Way # 4. Therapeutic Cloning:

This is a process for production of embryos for use in research by harvesting stem cells to treat human disease, also called “embryo cloning”. Stem cells have emerged as very important tools in biomedical research because they are potentially capable of producing any type of specialised cell in the human body. Stem cells are harvested from the blastocyst stage attained by the dividing egg in about five days. The embryo is destroyed in the process which raises ethical questions and concerns.