Plasmid DNA Isolation and Analysis, Research Paper Example

Introduction A plasmid is an extra-chromosomal genetic element which is capable of replicating on its own. It is a DNA molecule located externally to the bacterial chromosome and which   replicates autonomously and passes the molecules borne from replication to its daughter cells. Advances in DNA laboratory techniques which include DNA isolation, DNA fractionation, purification, DNA analysis, sequencing and amplification through the polymerase chain reaction (PCR) have contributed to the recent advances in gene manipulation called genetic engineering. Different methods of DNA isolation have been employed .These include alkaline lysis method used to purify plasmid DNA from bacterial cultures  using  organic solvents like chloroform, isoamyl alcohol, phenol . Another traditional DNA separation tool is the cesium chloride density gradient centrifugation, the lesser used technique which uses heat to extract plasmid DNA as well as the recently developed protocols which use special silica-based matrices to isolate plasmid DNA and at the same time concentrate this DNA.

During these extraction techniques proteins are denatured in the organic solvents and settle at the interface between the aqueous and organic phases.  . Most of these methods are tedious and time consuming because of the many steps involved and as a result advancements in DNA separation methods have been made making gene splicing possible. With the new improved methods it has been possible to separate DNA of different types and sizes within a shorter time. Such methods as the agarose gel electrophoresis methods have been developed in which  better results are achieved by first removing proteins using one of the other isolation techniques and then  further separations by electrophoresis.  Using gel electrophoresis is convenient because small fragments of DNA can be effectively separated within a very short time varying from 24 hrs to as little as 30 minutes. In addition the apparatus and reagents used in these procedures are cheap compared to the purchase and upkeep of an ultracentrifuge. A key to many of the procedures of DNA isolation is the discovery and use of restriction endonucleases produced by bacteria that degrade foreign DNA .The use of restriction enzymes has a biochemical significance in that they recognize unique nucleotide sequences and cause breaks only at these points and can therefore be used as a “molecular scalpel” excising specific regions of DNA. By cleaving DNA only at specific sites, sequences of interest to the researcher can be excised and used in another vector. Restriction Fragment Length Polymorphism (RFLP) technique can then be used to compare the genetic differences among the DNA of individuals within a population.

The main objective of this experiment is to isolate DNA from the extra-chromosomal DNA called plasmid thereby purifying only DNA. The recently developed filter mini-prep method will be employed in isolation and then DNA will be analyzed by agarose gel electrophoresis after digestion with restriction enzymes.

Discussion

The in vitro analysis and manipulation of plasmid DNA comes after an isolation step which frees the nucleic acid from cellular contaminants. Plasmid DNA was generated from a mini-scale sample preparation obtained from an overnight bacterial culture and after the isolation step agarose gel electrophoresis was run .The Plasmid DNA molecules from the lysate migrated on the Agarose gel as single bands at a rate inversely proportional to their molecular weights.

In the experiment the reagents used to separate plasmid DNA from bacterial RNA, DNA and proteins employ the principle of using three buffers in a sequence. Each of these buffers has unique compositions to help separate and concentrate plasmid DNA.  The first buffer resuspends the bacterial pellet got from an initial centrifugation step of the bacterial culture. After resuspension, buffer II whose components   (NaOH and SDS detergent) lyse the bacteria and denature the genomic DNA is added. Buffer III causes further denaturation of protein and also enhances binding of plasmid DNA to the silica matrix used in mini-prep columns. In addition the third Buffer also has potassium acetate which rapidly neutralizes the mixture of solutions.

Conclusion

The isolation and sizing of plasmid DNA has a broad application in molecular biology. Such applications include genomics, PCR products quality control and development of therapeutic drugs and vaccines which are genome-based .They are also widely used in genetic engineering as vectors to carry foreign DNA enabling its amplification isolation or expression. This experiment helped in understanding and getting hands on experience in DNA isolation and analysis techniques especially Agarose gel electrophoresis (AGE) being the core method for assessing the homogeneity of plasmid DNA. It also helped in understanding the role of restriction enzymes all which is very vital in molecular work.

Works Cited

Buyer, R.F. “Modern Experimental Biochemistry” Addison-Wesley publishing Co., Inc.

Lehninger, A., Nelson, D., Cox, M. “Principles of Biochemistry” 2^nd edition. Worth Publishers, Inc. 1993;211-222.

Norman-Tiner, C.  Biochemistry Lecture. Summary handout.