Cloning & DNA Recombination

    Cloning is one of the technologies in genetic engineering that allows humans to make duplicates both on a molecular scale and even on an individual scale. Cloning is a technique of multiplying a gene sequence (DNA) by combining a living thing's DNA sequence with the DNA of another living thing. The application of cloning to animals and plants has answered many questions in basic biology (Reece et.al., 2014). DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount. Steps in cloning a single piece of DNA are:

1. Appropriate restriction sites

2. Cut vector and foreign DNA with RE

3. Run on gel to separate fragments

4. Isolate specific fragment

5. Ligate with cut vector

6. Transform host bacteria. Selection.

7. Grow up colonies.

8. Isolate plasmid DNA.

9. Cut with RE to confirm presence of foreign DNA.

10. Run on gel to identify recombinant plasmids.

Recombination DNA is an attempt to put DNA from an organism into bacterial DNA by combining two or more sequences through gene splicing. Recombined DNA can occur naturally and artificially. Naturally, DNA recombination occurs through crossing over, transduction and transformation. Recombined DNA can be obtained artificially, namely by in vitro cutting and splicing. The stages in the DNA recombination process are: DNA isolation, cutting DNA strands using restriction endonuclease enzymes, splicing using ligase enzymes, DNA grafting into plasmid vectors, and inserting DNA into the host.

The target DNA fragment to be reproduced can be obtained by isolating the genome or isolating the mRNA as a substrate for reverse transcription to form complementary deoxyribonucleic acid (cDNA). Genomic DNA isolation was performed using standard PCR (polymerase chain reaction) technique and cDNA was obtained using RT PCR (real time polymerase chain reaction) technique. After the DNA fragments are obtained, the cloning vector is prepared so that the target DNA can be inserted. The most commonly used cloning vector is the plasmid. In recombinant DNA construction, the circular plasmid is cut (restricted) with a restriction endonuclease enzyme, then the target DNA is inserted and ligated into the plasmid (Susmiarsih, 2018).

Source: Susmiarsih (2018)

References:

Reece, J. B., Urry, L. A., Cain, M. L. 1., Wasserman, S. A., Minorsky, P. V., Jackson, R., & Campbell, N. A. 2014. Campbell Biology (Tenth edition). Boston: Pearson

Susmiarsih, T. P. 2018. Kajian DNA Rekombinan pada Vaksin DNA dan Vaksin Subunit Protein. Majalah Kesehatan PharmaMedika Vol. 10, No. 2: 108-128