Difference Between Gene Cloning and PCR

Key Difference – Gene Cloning vs PCR
 

The synthesis of many copies of DNA from a specific DNA fragment is called DNA amplification. There are two main DNA amplification processes namely gene cloning and PCR. The key difference between gene cloning and PCR is, gene cloning produces the multiple copies of a specific gene in vivo by constructing a recombinant DNA and growing inside a host bacterium while PCR produces millions of copies of a specific DNA fragment in vitro undergoing repeated cycles of denaturation and synthesis.

CONTENTS
1. Overview and Key Difference
2. What is Gene Cloning
3. What is PCR
4. Side by Side Comparison – Gene Cloning vs PCR
5. Summary

What is Gene Cloning?

Gene cloning is a technique employed to locate and multiply a specific gene from the extracted genomic DNA of an organism through the construction of recombinant DNA. Genomic DNA contains thousands of different genes encoded for proteins. When DNA is extracted, it includes all possible genes it can bear. Gene cloning technique has enabled the detection of a specific gene from the total DNA. Therefore gene cloning serves as an important tool in molecular biology.

Making of a genomic library of an organism is essential in gene cloning if there is no clue about the location of the relevant gene in the DNA. A genomic library is made using the following steps.

Step 1: Extraction of the total DNA from an organism which contains the desired gene.

Step 2: Restriction digestion of the extracted DNA to produce small manageable fragments. This step is facilitated by restriction endonucleases.

Step 3: Selection of a suitable vector and opening the vector DNA using the same restriction endonucleases. Bacterial plasmids are commonly used as vectors to carry foreign DNA. Plasmids are small circles of DNA located within bacteria.

Step 4: Combining of the vector DNA and fragmented DNA to produce recombinant DNA molecule. This step is governed by DNA ligase.

Step 5: Transferring of recombinant DNA molecules into host bacteria. This step is known as transformation, and is done using a heat shock.

Step 5: Screening of transformed bacterial cells on a culture medium. A mixed population of transformed and nontransformed host cells is obtained at the end of the transformation process. As gene of interest includes only in transformed host cells. Hence, it is necessary to select transformed cells. The selection is made using selective media which contain antibiotics. Only the transformed cells grow on this screening medium enabling the selection.

Step 6: Growing of bacteria to produce a gene library. In this step, the transformed host cells are introduced into fresh culture media which provides optimum growth requirements. Total colonies on the culture plates represent the genomic library of that organism.

Step 7: The recombinant DNA molecule containing the gene of interest must be screened from thousands of cloned fragments of recombinant DNA. It can be accomplished by the use of probes which mark the specific gene or the specific protein results from that gene.

Once the interested gene containing the bacterial colony is identified from the total colonies, it is possible to make millions of copies of the recombinant plasmid that contains the gene.

Gene cloning is used in establishing gene libraries, producing special protein, vitamins, antibiotics, hormones, sequencing and mapping genomes of the organisms, making multiple copies of individuals DNA in forensics etc.

What is PCR?

Polymerase Chain Reaction (PCR) is a technique which generates a large number of copies of a particular DNA fragment. Exponential amplification of a specific DNA sequence is obtained by PCR under in vitro conditions. This technique is a very powerful tool in Molecular Biology since it can multiply a small sample of DNA into a usable amount. PCR was introduced by Kary Mullis in 1983 and this prize-winning invention created a huge advancement in Molecular Biology.

PCR technique follows repeated PCR reactions as shown in Figure 02. One PCR reaction consists of three main steps occurring at three different temperatures; denaturing of double stranded at DNA at 94 ⁰C, annealing of primers at 68 ⁰C and strand elongation at 72 ⁰C. Therefore, when PCR is performed, temperature fluctuation should be highly maintained for proper replication. PCR is performed in a PCR machine inside PCR tubes. PCR tubes are loaded with correct PCR mixtures containing template DNA, Taq polymerase, primers, dNTPs and buffer. Denaturing of double stranded sample DNA into single stranded DNA is done by breaking the hydrogen bonds between complementary bases at 94 – 98 ⁰C. Then single strands of template DNA are exposed for primers. A pair of primers (forward and reverse) should be provided, and they should be thermostable to tolerate high temperatures. Primers are single stranded short DNA sequences complementary to ends of the target DNA fragment. Synthetic primers are used in PCR. Primers bind with the complementary bases of sample DNA and initiate the synthesis of a new strand. This step is catalyzed by an enzyme called Taq polymerase; a thermostable DNA polymerase enzyme isolated from Thermus auqaticus. When primers and nucleotides (building blocks) are available, Taq polymerase constructs the new strand of DNA complementary to template DNA. At the end of the PCR program, amplified DNA fragment is observed using gel electrophoresis. If further analysis is required, PCR product is purified from the gel.

PCR is very useful for diagnosing  and monitoring of genetic and acquired diseases, identification of criminals (in the field of forensics), studying the structure and function of a targeted segment of DNA, sequencing and mapping of genomes of organisms, etc. PCR has become a routine laboratory technique in medical and molecular biology research laboratories among scientists since it has a wide variety of applications.

What is the difference between Gene Cloning and PCR?

Summary – Gene Cloning vs PCR

Gene cloning and PCR are two methods used for DNA amplification. PCR is an in vitro process which makes multiple copies of DNA of a particular DNA fragment without using recombinant DNA and a host organism. Gene cloning is primarily an in vivo process which results in multiple copies of an interested gene inside the host organism via construction of recombinant DNA. This is the difference between Gene cloning and PCR.

Reference:
1. Griffiths, Anthony JF. “Cloning a Specific Gene.” Modern Genetic Analysis. U.S. National Library of Medicine, 01 Jan. 1999. Web. 22 Feb. 2017
2.”Polymerase Chain Reaction (PCR).” National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 22 Feb. 2017

Image Courtesy:
1. “Figure 17 01 06” By CNX OpenStax – (CC BY 4.0) via Commons Wikimedia
2. “PCR” By Madprime – Own work (CC BY-SA 3.0) via Commons Wikimedia