Overview
The DNA which contains DNA from two different sources is Recombinant DNA and the technology for the formation of this recombinant DNA is called recombinant DNA technology.
There are four requirements for recombinant DNA technology. First to get the gene that is to be cloned; called the gene of interest. Second molecular scissors are enzymes to cut the gene of interest. The third is a vector; carrier of recombinant DNA.
Gene can be synthesized in the lab to form mRNA using reverse transcriptase.
The DNA molecule is called complementary DNA (cDNA). Palindromic sequences are sequences of four or six nucleotides arranged symmetrically in the reverse order produced by restriction enzymes that cut the DNA at the specific site.
- 1) What is Recombinant DNA Technology
- 2) How to get a gene?
- 3) Molecular Scissors: Restriction Endonucleases
- 4) Molecular Carrier: Vector
- 5) Recombinant DNA
- 6) Expression of the Recombinant DNA
- 7) MCQs for Recombinant DNA Technology
- 8) Frequently Asked Questions (FAQs) on Recombinant DNA Technology
- 9) Summary: Recombinant DNA Technology
- 10) You may also like to learn:
What is Recombinant DNA Technology
Recombinant DNA technology popularly called genetic engineering targets synthesizing recombinant DNA which contains DNA from two different sources. In order to produce recombinant DNA, the following are needed:
- Gene of interest, which is to be cloned.
- Molecular scissors to eliminate the gene of interest.
- Molecular carrier or vector, on which gene of interest could be positioned.
- The gene of interest along with the vector is then introduced into an expression system, as a result of which a specific product is made.
How to get a gene?
There are three possible methods to get the gene of interest.
- (a) to isolate it from the chromosome
- (b) to manufacture it chemically, and
- (c) to make it from mRNA
Genes can be separated from the chromosomes by cutting the chromosomes on the flanking sites of the gene using special enzymes called restriction endonucleases. If, however, the genes are small, they can also be synthesized in the laboratory Another really typical technique of getting the gene is to synthesize it in the laboratory from messenger RNA, using reverse transcriptase. This DNA molecule is called complementary DNA (cDNA).
Molecular Scissors: Restriction Endonucleases
These are natural enzymes of bacteria, which they use for their own defense against viruses. The restriction enzyme cuts the viral DNA, however does no harm to the bacterial chromosome. They are called restriction enzymes because they limit the growth of infections. In 1970, Hamilton O. Smith, at Johns Hopkins University, isolated the first restriction enzyme.
Bacteria produce a range of such restriction enzymes, which cut the DNA at extremely specific sites characterized by a specific sequence of 4 or 6 nucleotides organized symmetrically in the reverse order. Such series are called palindromic series. So far more than 400 such enzymes have actually been separated out of which about 20 are regularly used in recombinant DNA technology.
EcoRl, a commonly used restriction enzyme, cuts double-stranded DNA when it has this series of bases at the cleavage site. Observe there is now space into which a piece of foreign DNA can be put if it ends in bases complementary to those exposed by the restriction enzyme. The single-stranded but complementary ends of the two DNA molecules are called “sticky ends” since they can bind by complementary base pairing. They, for that reason, assist in the insertion of foreign DNA into vector DNA.
Molecular Carrier: Vector
To make recombinant DNA, one frequently begins by selecting a vector, the means by which recombinant DNA is introduced into a host cell. One common type of vector is a plasmid. Plasmids were discovered by investigators studying the sex life of the digestive tract bacterium Escherichia coli.
Plasmids are natural extra-chromosomal circular DNA molecules that carry genes for antibiotic resistance and fertility etc. One of the plasmids found earlier pSC 101 has an antibiotic resistance gene for tetracycline, whereas pBR 322 has antibiotic resistance genes for tetracycline along with ampicillin. Placing a gene of interest in the tetracycline-resistant gene of plasmid pBR 322 would allow separating out colonies of bacteria in a medium including ampicillin and vice versa.
Recombinant DNA
For the preparation of recombinant DNA, the plasmid is cut with the same enzyme which was used for the isolation of the gene of interest. The gene of interest (insulin) is then accompanied by the sticky ends produced after cutting the plasmid with the help of another special enzyme known as DNA ligase.
This enzyme seals the foreign piece of DNA into the vector. Now the two different pieces of DNA have been joined together, which is now called recombinant DNA or chimeric DNA.
Expression of the Recombinant DNA
A clone can be a large number of particles (i.e., cloned genes) or cells (i.e., cloned bacteria) or organisms that are identical to an initial specimen. Bacterial cells take up the recombinant plasmid, specifically, if they are treated with calcium chloride to make them more permeable. Afterward, as the cell recreates, a bacterial clone kind and each new cell consist of at least one plasmid.
For that reason, each of the bacteria contains the gene of interest, which will express itself and make a product. From this bacterial clone, the cloned gene can be isolated for further analysis, or the protein product can be separated. Besides plasmids, the DNA of bacterial viruses (for instance, lambda phage) can likewise be utilized as a vector.
After the lambda phage attaches to a host bacterium, recombinant DNA is released from the virus and gets in the bacterium. Here, it will direct the reproduction of many more viruses. Each virus in the bacteriophage clone consists of a copy of the gene being cloned.
MCQs for Recombinant DNA Technology
- What is Recombinant DNA?
- a) DNA from a single source
- b) DNA from two different sources
- c) DNA from bacteria only
- d) RNA from animals
Answer: b
- Which technology is used for the formation of Recombinant DNA?
- a) Cloning technology
- b) Genetic engineering
- c) Reproductive cloning
- d) Therapeutic cloning
Answer: b
- What are the four requirements for Recombinant DNA technology?
- a) Gene, Vector, Enzyme, Protein
- b) Gene, Vector, RNA, Expression System
- c) Gene, Enzyme, Plasmid, Expression System
- d) Gene, Enzyme, Vector, Expression System
Answer: d
- How can a gene of interest be obtained?
- a) Isolate it from the chromosome
- b) Manufacture it chemically
- c) Make it from mRNA
- d) All of the above
Answer: d
- What is cDNA?
- a) Complementary RNA
- b) Circular DNA
- c) Cloned DNA
- d) Complementary DNA
Answer: d
- What are palindromic sequences in DNA?
- a) Sequences that read the same backward as forward
- b) Sequences that code for proteins
- c) Sequences involved in mRNA synthesis
- d) Sequences found only in bacteria
Answer: a
- Who isolated the first restriction enzyme?
- a) James Watson
- b) Francis Crick
- c) Hamilton O. Smith
- d) Gregor Mendel
Answer: c
- What do restriction enzymes do in bacteria?
- a) Limit bacterial growth
- b) Limit viral growth
- c) Limit gene expression
- d) Limit RNA synthesis
Answer: b
- What is the role of EcoRI in DNA technology?
- a) Synthesizes DNA
- b) Cuts double-stranded DNA at specific sites
- c) Forms recombinant DNA
- d) Converts RNA to DNA
Answer: b
- What is a common type of vector used in DNA technology?
- a) mRNA
- b) Plasmid
- c) tRNA
- d) rRNA
Answer: b
- What is the purpose of DNA ligase in recombinant DNA preparation?
- a) Cuts DNA
- b) Synthesizes DNA
- c) Seals foreign DNA into the vector
- d) Breaks down DNA
Answer: c
- What is a clone in the context of recombinant DNA technology?
- a) A gene
- b) A virus
- c) Identical particles, cells, or organisms
- d) A restriction enzyme
Answer: c
- How can a bacterial clone be created in DNA technology?
- a) By isolating genes
- b) By using reverse transcriptase
- c) By treating cells with calcium chloride
- d) By synthesizing mRNA
Answer: c
- What is the role of a plasmid in recombinant DNA technology?
- a) Transcribes genes
- b) Carries antibiotic resistance genes
- c) Synthesizes mRNA
- d) Isolates genes from chromosomes
Answer: b
- Which vector, besides plasmids, can be used in DNA technology?
- a) mRNA
- b) tRNA
- c) rRNA
- d) Bacterial viruses (e.g., lambda phage)
Answer: d
Frequently Asked Questions (FAQs) on Recombinant DNA Technology
- What is Recombinant DNA?
- Answer: Recombinant DNA refers to DNA that contains genetic material from two different sources. It is formed using the technology known as recombinant DNA technology.
- What are the four requirements for recombinant DNA technology?
- Answer: The four requirements are:
- Gene of interest (to be cloned)
- Molecular scissors (enzymes to cut the gene of interest)
- Vector (carrier of recombinant DNA)
- Expression system
- Answer: The four requirements are:
- How can a gene be obtained for cloning?
- Answer: Genes can be obtained by isolating them from the chromosome, manufacturing them chemically, or synthesizing them from mRNA in the laboratory.
- What are palindromic sequences in DNA, and why are they important?
- Answer: Palindromic sequences are sequences of four or six nucleotides arranged symmetrically in the reverse order. They are recognized by restriction enzymes and are crucial in cutting DNA at specific sites.
- What is the role of restriction endonucleases (molecular scissors) in recombinant DNA technology?
- Answer: Restriction endonucleases, natural enzymes of bacteria, cut DNA at specific sites. They are used to eliminate the gene of interest from the chromosome.
- How are vectors used in recombinant DNA technology?
- Answer: Vectors, such as plasmids, serve as carriers for introducing recombinant DNA into host cells. Plasmids are circular DNA molecules that can replicate independently.
- What is the significance of “sticky ends” in recombinant DNA technology?
- Answer: Sticky ends are single-stranded complementary ends produced after cutting DNA with restriction enzymes. They facilitate the insertion of foreign DNA into vector DNA.
- What is the purpose of DNA ligase in recombinant DNA preparation?
- Answer: DNA ligase seals the foreign piece of DNA into the vector by joining the sticky ends, resulting in the formation of recombinant DNA.
- Can bacterial viruses be used as vectors in recombinant DNA technology?
- Answer: Yes, bacterial viruses, such as lambda phage, can be used as vectors. They release recombinant DNA into the host bacterium, directing the reproduction of more viruses.
- How is the expression of recombinant DNA achieved?
- Answer: Recombinant plasmids are introduced into bacterial cells, forming bacterial clones. As the cells reproduce, each new cell contains at least one plasmid, expressing the gene of interest and producing the desired product.
- What is a clone in the context of recombinant DNA technology?
- Answer: A clone can refer to a large number of particles (genes), cells (bacteria), or organisms that are identical to the original specimen. In recombinant DNA technology, bacterial clones are common.
- Which commonly used restriction enzyme cuts double-stranded DNA at specific sites?
- Answer: EcoRI is a commonly used restriction enzyme that cuts double-stranded DNA at specific sites with a palindromic sequence.
- How are recombinant DNA and chimeric DNA related?
- Answer: Recombinant DNA, formed by joining the gene of interest with a vector, is also known as chimeric DNA.
- What is the significance of treating bacterial cells with calcium chloride in recombinant DNA technology?
- Answer: Treating bacterial cells with calcium chloride makes them more permeable, allowing them to take up recombinant plasmids efficiently.
- Besides plasmids, what other types of vectors can be used in recombinant DNA technology?
- Answer: Besides plasmids, the DNA of bacterial viruses (e.g., lambda phage) can also be used as vectors in recombinant DNA technology.
Summary: Recombinant DNA Technology
Recombinant DNA technology involves the creation of DNA molecules that combine genetic material from two different sources. The process encompasses several key steps and components.
- Overview:
- Recombinant DNA is formed by combining DNA from distinct sources, and the technology enabling this process is known as recombinant DNA technology.
- The essential components for this technology include the gene of interest (to be cloned), molecular scissors (enzymes for gene cutting), a vector (DNA carrier), and an expression system.
- Gene Acquisition:
- Genes can be obtained through three methods: isolation from the chromosome, chemical synthesis, or laboratory synthesis from mRNA.
- Palindromic sequences, recognized by restriction endonucleases, play a crucial role in cutting DNA at specific sites.
- Molecular Scissors – Restriction Endonucleases:
- Bacterial enzymes, known as restriction endonucleases, act as molecular scissors to cut DNA without harming the bacterial chromosome.
- Over 400 restriction enzymes, with palindromic recognition sequences, have been identified, including the commonly used EcoRI.
- Molecular Carrier – Vector:
- Vectors, such as plasmids, are used to introduce recombinant DNA into host cells.
- Plasmids, circular DNA molecules, carry genes and are commonly employed in recombinant DNA technology.
- Recombinant DNA Formation:
- Recombinant DNA is prepared by cutting the plasmid with a specific enzyme, accompanied by the gene of interest (e.g., insulin), and sealed with DNA ligase.
- The resulting combination is referred to as recombinant DNA or chimeric DNA.
- Expression of Recombinant DNA:
- Bacterial cells take up recombinant plasmids, forming bacterial clones when treated with calcium chloride.
- Each bacterium in the clone contains the gene of interest, expressing itself to produce a specific product.
- Besides plasmids, bacterial viruses (e.g., lambda phage) can serve as vectors for recombinant DNA.
In conclusion, recombinant DNA technology offers a powerful means to manipulate and utilize genetic material, enabling the expression of desired genes and the production of specific gene products.