QUALITATIVE AND QUANTITATIVE ANALYSIS OF DNA BY SPECTROPHOTOMETRY

 

RESULTS
The PCR primers were optimized for the annealing temperatures, MgCl2 concentrations and template concentrations. The primers were standardized at a range (54degC- to 60degC) of annealing temperatures. The result was given in table (2). The amplification was good at 58degC for all the primers. The MgCl2 concentrations of 1.5mM and 2.5mM were used to standardize the primers. The result was given in the table (3). All the products were amplified at both the MgCl2 concentrations. Low MgCl2 concentration was used in subsequent analysis. The template concentrations chosen for the amplification were 100 ng and 150 ng. The result was given in the table (4). After the amplification of the individual fragments with a volume of 50 ul each were pooled together and precipitated with 100% ethanol. After the ethanol precipitation the pellet was resuspended in 100 ul of the nuclease free water and 3 ul of the sample was loaded on the gel along with the reference molecular weight marker from MBI fermantas. The concentrated molecular weight marker and the reference marker were shown in the Figure (4). 

All the fragments intensity was good when compare to reference marker and the resolution between the marker bands between the home maid and commercial molecular weight markers were same.

S.NO

Primer sequence

1

F- 5’-TCGCGCGTTTCGGTGATGAC-3’

2

1R- 5’- CGGGCTTGTCTGCTCCCGGC-3’

3

2R-5’-TATTTCACACCGCATATGGT-3’

4

3R-5-’ATACGAAGAGGCCCGCACC-3’

5

4R-5’-AATTCACTGGCCGTCGTTTT-3’

6

5R-5-’TGGAATTGTGAGCGGATAAC-3’

7

6R-5-’GGGCAGTGAGCGCAACGCAA-3’

8

7R-5- ATTAATGCAGCTGGCACGAC-3’

9

8R-5-’CCTGCGTTATCCCCTGATTG-3’

10

9R-5-GTGATGCTCGTCAGGGGGGC-3’

11

10-5’-TCGGAACAGGAGAGCGCACG-3’

Table1: The sequence of the primers used for the amplification of the different fragments in the molecular weight marker

Primer

Annealing  temperature

Result

Annealing  temperature

Result

Annealing  temperature

Result

Annealing temperature

Result

MF+MR1

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR2

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR3

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR4

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR5

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR6

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR7

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR8

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR9

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

MF+MR10

54degC

No amplification

56degC

No amplification

58degC

Good amplification

60degC

No amplification

Table2: Different annealing temperatures and the amplification of the PCR product

Primer

Annealing temperature

MgCl2 concentration

Result

MgCl2 concentration

Result

MF+MR1

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR2

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR3

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR4

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR5

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR6

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR7

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR8

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR9

58degC

1.5mM

Good amplication

2.5mM

Good amplication

MF+ MR10

58degC

1.5mM

Good amplication

2.5mM

Good amplication

Table3: Different MgCl2 concentrations and the result of the PCR  amplification

Primer

Annealing temperature

MgCl2 concentration

Template concentration

Result

Template concentration

Result

MF+MR1

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR2

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR3

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR4

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR5

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR6

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR7

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR8

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR9

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

MF+MR10

580 C

1.5mM

100ng/µl

Less amplification

150ng/µl

Good amplication

Table4: Different templateconcentrations and the result of the PCR amplification.

DISCUSSION The molecular weight markers are very important tools in molecular biology and in protein science. Any nucleic acid molecule to be sized should run along with known size standards. The known size standards available in 50ug and 250ug. That can be used for 220 and 1100 applications respectively. The price in market is ranging from 3000 rupees to 6000 rupees depends upon the company. Per application it costs around 15-30 rupees. If we can synthesize the marker in the laboratory the price will be 5rupees for application. To minimize the price we had synthesized the molecular weight marker in our laboratory. The marker constructed in the laboratory had the entire bands as commercially available marker. Higher molecular weight bands had low intensity that could be due to the low recovery of the DNA at the time of precipitation. precipitation protocol has to standardize to get proper molecular weight  marker.

CONCLUSION Good intensified marker was developed by individual fragment amplification and pooling method. The resolution between all the fragments in the molecular weight marker was clear.

BIBLIOGRAPHY

  1.     Joseph Sambrook and David W. Russel (2001). Molecular Cloning: A Laboratory Manual (3rd ed. ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-87969-576-5.  Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
  2.     Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22: 253–260. doi:10.1016/j.tibtech.2004.02.011. PMID 15109812.
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  7.     Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes". in Kieleczawa J. DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett. pp. 241–257. ISBN 0-7637338-3-0.
  8.      Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343)". Arizona State University. photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/DNAtech.html. Retrieved on 2007-10-29.
  9. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, and Markham AF (1989). "Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)". Nucleic Acids Research 17 (7): 2503–2516. 

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how the DNA is diluted 100 times?

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