Determining the orientation of insert DNA

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Polymerase chain reaction (PCR) is a scientific technique used in molecular biology in manipulating small fragment of DNA into thousands to millions of copies of a particular DNA sequences in a process called amplification. The discovering of PCR development by Mullis in the 1980s has led to the ability of DNA manipulation in both medical and biological research labs for various applications; for example, many conventional diagnostic approaches to detection and characterisation of infectious diseases is complemented or rather replaced by recognition of DNA/RNA (deoxyribose nucleic acid or ribose nucleic acid) specific sequences. There are two distinct types of methods in this molecular processes; nucleic acid hybridization techniques with specific probes, and DNA amplification by PCR. The first method involved the use of restriction endonucleases that cuts DNA into fragments and separated by electrophoresis, then transferred to a proper membrane called vector and annealed with specific oligonucleotides, which are either radioactively label as 32P or without radioactive label .

The manipulation of a DNA and nucleic acid sequencing has produced remarkable advances in biochemistry, genetic and cell biology areas. The recombinant DNA know-how has made it achievable to purify specific target DNA sequences and amplify them for studies. The recombinant DNA technology also called molecular cloning (produce a multiple of identical organisms derived from a single ancestor) or genetic engineering has made it possible to isolate tiny quantities of DNA, amplify, and modify specific DNA sequences. Amplification is done through processes. A fragment of DNA of the suitable size is generated by a restriction enzyme, by PCR, or by chemical synthase. This fragment of DNA is then incorporated into a vector, which contains the sequences necessary to direct the replication of the interest DNA. The vector with the inserted DNA is introduced into a cell where it replicates. Cells containing the desired DNA are indentified and selected.

Plasmids are circular DNA molecules of 1 to 200 kilobases found in bacteria or yeast cells. Plasmids can be consider as molecular parasites, but benefited their hosts by providing functions, such as resistance to antibiotic, lacking from the host cell. Plasmids used for cloning are usually present in hundreds of copies to a huge number of thousand copies. The conventional laboratory plasmids are relatively small, replicate easily, and carry genes specifying resistance to more antibiotic, with convenient located restriction endonuclease sites for foreign DNA insertion. Some examples of clone vectors are E.Coil bacteria, bacteriophage, etc. Some larger vectors can hold clone DNA up to a number of hundreds kilobase pairs. These larger vectors are called bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs).

Ligase joins two DNA segment. As first demonstrated by Mertz and Davis in 1972, restriction fragment are inserted into the cut clone by restriction enzyme. The complimentary ends of the two DNA form base pair (anneal) and the sugar-phosphate backbones are covalently ligated through the DNA ligase. The advantage of using restriction enzyme is that, the same restriction enzyme can be use to insert plasmid DNA and later be precisely excised from the cloned vector by cleaving it using the same restriction enzyme.

Medical application

Gene mapping is a clinic diagnostic measures in several disease-relative genes for isolating using probes specific for nearby markers, such as recurring DNA sequences, that were previously identified to be genetically related to the disease genes.DNA is amplified by the PCR. Molecular cloning techniques are indispensable to modern biochemistry researches. The PCR is often a quicker and more suitable method for amplifying a precise DNA. A segment of up to 6kb can be amplified using this technique. In PCR, a DNA sample is split into distinct strands and incubated with DNA polymerase, dNTPs, and two oligonucleotides primers whose sequence flank the DNA of complementary DNA target by DNA polymerase. The following circle in PCR the products become templates for replication in a chain reaction whereby the DNA template is modified to perform a wide array of genetic manipulations such as clinical diagnose se infectious diseases and to identify atypical pathological measures such as mutations leading to Cancer. Forensically, the DNA from a single hair or Sperm can be amplified by PCR to identify the donor. PCR is effective on pinhead-size biological fluids samples, and Courts now accept PCR results to restored justice to convicts, even many years after the crime scene evidence had been collected.

In this experiment, we used PCR reaction to determine the orientation of the 246bp insert pure plasmid PCER-D DNA, determined from Experiment 5.1 and 5.2 with primers Ext-, X and Y.

Aims: To design and perform and experiment to determine the direction of an insert DNA 246bp within a pure plasmid (PCER-D DNA).

Safety notes: Bio1302 workbook P14-15 on safety notes and additional hazards where followed.

Choice of Primers

To determine the forward orientation of the 246bp plasmid pCER-D insert, primers Ext- and X were used as they will amplify within the insert hence giving details in a forward direction.

To determine the reverse orientation of the 246bp plasmid PCER-D insert, Primers Ext-and Y were used as they will amplify within the insert hence giving details in the reverse direction.

Expected Outcome

For the forward orientation, primers Ext- and X will successfully amplify plasmid pCER-D DNA within the insert, thereby giving 558bp ((1127-611) + 42).

For the reverse orientation, primers Ext- and Y will successfully amplify plasmid PCER-D DNA within the insert, thereby giving 742bp ((1127-611) + 226). Primers Ext+ and Ext- were not chosen even though they are complementary pairs, because they will amplify outside the insert plasmid PCER-D DNA, thus would not give any details about the orientation.

The choice of PCR over a restriction digestion method was not due to nor restriction site found on the cloned DNA, therefore not suitable in this experiment.

Method-PCR

Preparation method of samples for PCR in workbook BioC1302 P17-18 where followed. DNA sample A was used as templates for this experiment. PCR is a very sensitive process, and amplifies small amount of template exponentially to a very large quantity of DNA, each of the plasmids will be diluted by a factor of 1/10 in order to start a reaction. Two different loadings of product were loaded on the agarose gel electrophoresis to produce a result with certainty.

Firstly, sample A DNA from Experiment 5.1 was diluted 1/10 using sterile water.

Secondly, two PCR tubes were labelled AX and AY and kept on the ice whilst having the following added to them in order: 7.5µl (microlitres) of sterile H2O (water), 2 µl 10X PCR reaction buffer, 2 µl 15 mM MgCl2 (magnesium Chloride), 2 µl of stock 2 mM dNTP solution, 2 µl of 10 µM first primer X or Y (depending on the label tube), 2 µl if 10 µM second primer: Ext- (for both primer X and Y), 2 µl of diluted plasmid A as template, 2 µ MgCl2 (15mM), and 0.5 µl of diluted Taq polymerase (5U/µl) (added last).

The two tubes were closed and contents mixed with brief centrifugation to collect solution to the bottom of the tubes. Due to the small volumes of 2µl and less, we used P2 pipettes with small clear plastic sterile tips for accuracy in pipetting.

The completed pairs (Ext- -X and Ext- -y) were loaded in the thermal cycle (PCR machine) with the help of the demonstrator. The cycle was automatically circle under the following conditions: 95oC for 1' for initial denaturation of template, 25oC cycles of 95oC, 1' (denaturation) 50oC, 1' (primer annealing), 72oC, 2' (extension) 72oC for 10' (final extension of an incomplete fragment). This whole process lasted about 2.5 to 3 hours.

Agarose gel was prepared whilst waiting for the PCR to complete.

Preparing the agarose gel:

A 1.2g of agarose was weighed and transferred to 100mL beaker, then dissolved with 100mL of TAE electrophoresis buffer. This gave a final agarose concentration 1.2% (W/V), which is similar to experiment 5.2, due to the small fragment sizes. A magnetic stirrer was inserted in the beaker and covered with foil. The solution was melted under microwave at 30 second intervals until fully melted. The dissolved agarose was set aside to cool to 60oC by monitoring with thermometer. While waiting for the agar to cool to 60oC, a mould was prepared for the gel by sticking autoclave tape around the sides of an electrophoresis plate. A lever surface was used to position a slot former 1cm away from the edge and 1mm above the surface of the plate with chips.

After the agarose was cooled to 60oC, 20µl of 5mg/mL eithidium bromide solution was added. Wearing a pair of gloves, the solution was shirred and gently mixed then poured carefully into the gel mould to a depth of 6-7 mm without splashing. The gel was left to set for 30mins, before removing the slot former by easing it out from one end to avoid tear on the thin layer of the gel between the former and the glass plate. This was handled with thorough care. The gel was then transfer to the electrophoresis tank and added with enough TAE buffer covering 1mm gel surface, while still wearing a pair of gloves.

Once the PCR completes, 4µl of 6X sucrose dye mix was added to each tube, flick to mix and centrifuge for at least 30 seconds. The following were then added to the separate adjacent slots in the agarose gel: 3 µl DNA size marker (100bp ladder) in the first slot, 4 µl PCR sample AX in the second slot and 4µl PCR sample AY in the third slot. The fragments were separated by electrophoresis (10V/cm), allowing the gel to run for 90minutes. At this concentration of agarose the bromophonol blue dye in the loading buffer behaves like a band of 300bp, stopping the gel before it migrates off the bottom.

The agarose gel was then photograph after electrophoresis to measure the migration of the size marker bands, and constructed a calibration curve to determine the size of fragments X and Y. The resultant sizes of the fragments with the relative respective primers will indicate the orientation of the insert in the pCER-D plasmid DNA.

Results:

Well no 1: 100bp ladder

Well no 2: AX

Well no 3: AY

Title: Table 1 of molecular marker (100bp) with relative distance travelled (mm)

Size (100bp)

Log10 (100bp size)

Distance travelled (mm)

1,517

3.18

34

1,200

3.08

37

1,000

3.00

40

900

2.95

41

800

2.90

43

700

2.85

45

600

2.78

47

500/517

2.71

50

400

2.60

53

300

2.48

56

200

2.30

59

Title: Table 2 showing the number of bands on agarose gel of primers Ext- and X PCR products.

Number of band

Distance travelled (mm)

Log10(base pair)

Antilog10(base pair)

Ext- and X

1

48

2.74

550

Percentage accuracy: 550/558 x 100 = 98.57%

Discussion

We have indentified the orientation of the insert plasmid pCER-D in the forward direction, as this gives the product as the expected 558bp amplification. We could not find any band for the reversed orientation which was suppose to give 742bp amplification, this confirmed that the insert plasmid PCER-D was only in the forward direction. Table 2 show 550bp product deduced from the graph which has slight difference with the actual 558bp, this could be due to the resolution power of the gel that cannot resolved to the last base pair. The result achieved from the graph (550bp insert plasmid PCER-D) confirms the forward orientation of primers Ext- and X insert DNA amplification.

PCR depends on the primers and where they bind. In this experiment primer Ext- binds on the 1127bp while primer X binds on the 611bp therefore amplifying insert plasmid PCER-D DNA to 558bp.

100bp ladder in the agarose gel electrophoresis used due to small fragments we dealing with, as 1kb (kilo base) will not be able to give a fair distribution calibration curve compare to the 100bp which gives a fair distribution calibration curve. 4µl of PCR products (AX and AY) were used on the agarose gel to measure the migration of the size fragment from the calibration graph for accuracy as 20µl will produce a think band which will be not give an accurate measurement.

Although PCR is often the fastest and more convenient method of amplifying a specific DNA, its large sensitivity its main drawbacks since the very large degree of amplification makes the system vulnerable to contamination. Hence cleanliness is of paramount importance when carrying out PCR and dedicated equipment are necessary to avoid misleading results.

Conclusion

The aim of this experiment was to determine the orientation of the cloned insert 246bp within a pure plasmid pCER-D. By using primers Ext-, X and Y in the PCR amplification of the cloned insert through agarose gel electrophoresis, it was established that the cloned insert DNA was in the forward orientation (Ext- and X) of 550bp to the actual 558bp. AS the gel cannot resolved to the last base pair, the slight difference was due this factor when deducing from the calibrated graph.

The result achieved from the agarose gel shows only one band for primers Ext- and X (forward orientation) and no band for Ext- and Y (reverse orientation), which indicated that the cloned insert within the pure plasmid pCER-D was in forward direction only and therefore the reverse was the control in this experiment.

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