Sample Report On Polymerase Chain (Pcr) Amplification Of Cdna Inserts From Recombinant Plasmids

Introduction

Polymerase chain reaction (PCR) is a process that amplifies a selected region of DNA using oligonucleotides called primers. The selected region of DNA undergoes numerous cycles of denaturation, annealing, and extension, which doubles the number of amplicon each time. In each cycle, the temperature varies to allow each step to occur: (a) first, denaturation is achieved using high temperature to separate the two strands of the DNA; (b) second, the temperature is brought down to a specific temperature to allow the primers to bind to their recognized sites; and (c) third, the temperature is brought up to the optimal temperature for the DNA polymerase to extend the sequence using the primers as “anchors” or starting points. The cycle is repeated until a large amount, enough to be visualized through electrophoresis, has been made (Weaver 2008).
PCR is used extensively in molecular biology and has been modified to create various types that accommodate the various needs of research studies. The basic PCR reaction is a standard process in recombinant technology, especially for cloning specific genes which are then placed into specific vectors. PCR can also be used to screen colonies for positive transformants or to confirm if a plasmid obtained from a culture contains the desired insert.
In this experiment, PCR is used to amplify the cDNA inserts (Neil1, Neil2, and Neil3) of three recombinant plasmids (pGEM®-T, Promega) extracted and purified from a previous exercise. The main objective is to observe the different inserts and to distinguish them through their band sizes.

Methodology

A. Polymerase Chain Reaction (PCR)
The PCR reaction mixtures were prepared in three 0.2-mL microcentrifuge tubes. Each tube contained 5 µL of the plasmid (DNA sample), 5 µL of the forward primer (T7), 5 µL of the reverse primer (Sp6), 10 µL distilled water (dH2O), and 25 µL of the "MyTaq Red Mix" (Bioline) premix. The samples were loaded into a thermocycler machine programmed with the following profile:

B. Agarose Gel Electrophoresis (AGE) of the PCR samples

Three 20-µL samples were prepared by mixing 5µL of each PCR reaction mix, 5µL of loading dye, and 10 µL of dH2O in 0.5-mL tubes.
A 0.8% agarose gel was prepared by dissolving 0.24 g of agarose in a conical flask with 30mL 0.5X Tris-borate-EDTA (TBE) buffer. The mixture was heated in a microwave in 30-second bursts and swirled until the agarose has fully dissolved, and left to cool to 55C. Thirty microliters (30 µL) of GelRed (1:1000 dilution) was then added to the liquid agarose. The gel mixture was poured into a tray, the comb was added, and was left to set for 30 minutes.
Once the gel has set, the comb was removed and the gel tray was placed in the electrophoresis chamber, with the wells closer to the negative electrode. The chamber was then filled with 0.5X TBE. Ten µL of the DNA size marker, Hyperladder I, was loaded into the first well of the gel. For the PCR reactions, the 20-µL samples were mixed with loading buffer and 10 µL of each were loaded into three separate wells. The gel was run at 100V for 40 to 45 minutes and viewed using a UV-transilluminator.

Results

Figure 1. Agarose gel electrophoresis of PCR amplified inserts. (MW) – molecular weight marker (HyperLadder I); (1) – Neil1; (2) – Neil2; (3) Neil3

Discussion

PCR amplification of DNA fragments is an invaluable technique used in the field of molecular biology. The basic protocol has been improved upon and modified to suit the purposes of various studies. The first step of the protocol involves the preparation of the reaction mixtures, which include the template DNA, forward and reverse primers, deoxyribonucleoside triphosphates (dNTPs), DNA polymerase/s, PCR buffer, and water. The reactions are loaded into a thermocycler which regulates the temperature of the PCR process (Ausubel et al. 2003).
In this experiment, the pGEM®-T plasmids which contained cloned Neil1, Neil2, and Neil3 genes served as the DNA template. These recombinant plasmids were extracted and purified in a previous experiment. In order to amplify these three different genes at the same time, universal primers (T7 5’-TAATACGACTCACTATAGGG-3’; and Sp6 5’-ATTTAGGTGACACTATAG-3’) were used. Primers are necessary in PCR because the DNA polymerase cannot start replication from scratch—it can only add to a free 3'-hydroxyl group of a growing or existing strand attached to a template (Berg, Tymoczko, & Stryer 2002). Primers are also usually specific, which allows the researcher to amplify the targeted or desired DNA fragment.
The pGEM®-T plasmid contains the T7 and Sp6 promoters that flank the multiple cloning site of the plasmid. These are promoter regions that are binding sites of RNA polymerase, thereby enabling transcription and expression of the cloned gene in an appropriate expression system. Their convenient location also allows them to be reference points for sequencing (directional) and PCR amplification of the insert. It is important to note that while the pGEM®-T plasmid has T7 and Sp6 promoters it is a cloning vector and is recommended to be used as an expression vector (Promega Corporation 2010).
PCR amplification of a recombinant plasmid is one way of confirming or identifying its contents without having to sequence the insert. Another way to confirm a plasmid’s insert is to use restriction enzymes (REs), and this was performed in the previous experiment. Both techniques involve visualizing the bands through agarose gel electrophoresis (AGE). It is also necessary to know beforehand the expected length or size of the bands in base pairs (bp).
Since the inserts were appended with NdeI and HindIII restriction sites, the data from the double digest should yield a more accurate estimation of the insert lengths. Moreover, the calculated band sizes for the PCR products should be slightly larger than the calculated sizes of the RE-digested fragments. This is because the T7 and Sp6 regions flank the multiple cloning site of the plasmid, which would be amplified by PCR as well, adding about 130 base pairs more than the insert’s actual length. However, the slight difference could be hard to distinguish using AGE, because it is a qualitative analytical technique, not a quantitative one.
Visual analysis of the PCR amplified inserts and the RE-digested (NdeI + HindIII double digest) fragments both show similar profiles in the AGE photos, in terms of the band locations relative to the DNA ladder. The band sizes were also calculated by plotting the log10 size (bp) of the molecular weight marker (HyperLadder I) bands against their migration distance from the well, using the software Microsoft Excel 2010. Since the relationship is known to be linear (also inverse), trendline analysis of the data yielded a line equation that was used to estimate the sizes of the inserts using their migration distance in the gel. Alternatively, linear regression analysis of the data also yielded the same results. Thus, using the gel from the previous experiment, the cDNA inserts sizes were estimated to be 1392 bp, 1182 bp, and 2157 bp for Neil1, Neil2, and Neil3, respectively. Meanwhile, using the gel profile of the PCR products, the calculated lengths were 1472 bp (Neil1), 1194 bp (Neil 2), and 2086 bp (Neil3) (refer to Table 1).
The calculated band sizes from both experiments were roughly the same, as was expected. The calculated sizes of the PCR amplicons (except for Neil3) were also bigger than the RE-digested inserts, as was expected as well. The discrepancy for the Neil3 calculated size (i.e. PCR amplicon being smaller than the RE-digested insert) can be due to the fact that the gel for this experiment was poorly resolved (as compared to the RE digestion gel) and negatively affected the calculation.
In summary, PCR amplification of Neil1, Neil2, and Neil3 cDNA inserts from recombinant pGEM®-T plasmids was successful. The three inserts were observed to have different lengths through AGE analysis. The amplicons also displayed a similar gel profile to that of the double-digested (RE-released) inserts from the previous experiment. The PCR amplified genes were calculated to have bigger sizes than their RE-digested counterparts, except for Neil3 (attributable to poor gel resolution). In order to more effectively visualize the differences in size between the RE-digested and PCR-amplified inserts, reactions from both experiments can be electrophoresed side by side in a gel of higher agarose concentration.

References

Ausubel FM, Brent R, Kingston RE, et al. 2003, Cuurent Protocols in Molecular Biology, John Wiley & Sons, USA.
Berg JM, Tymoczko JL, & Stryer L 2002, Biochemistry, 5th edn, W.H. Freeman, NY
Promega Corporation 2010, pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual, viewed 15 February 2015, from www.promega.com/protocols/
Weaver, RF 2012, Molecular Biology, 5th edn, The McGraw-Hill Companies, NY.