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Laboratory Report III:

DNA CHARACTERIZATION

 

 

Basics in Biology KMB015, Autumn 2004

(MSc in Bioinformatics)

Examiner: Markus Tamás

 

 

Author: Yoana Dimitrova
Date:
September 25, 2004

 

Lab partners: Yoana Dimitrova,

                  Robert Kirchner

   



Introduction

 

Deoxyribonucleic acid (DNA) is the carrier of the genetic material and as such, it predetermines the biochemical life of the cell. It is not surprising that DNA characterization has become an important tool in every biological laboratory.

 

There are various methods for DNA analysis and two of the most popular were used in this laboratory practical. The first one takes advantage of restriction nucleases which have the important property of slicing DNA in specific locations. The restriction enzyme used in this practical is called KpnI and the target sequence was in the pYX212-DAK1 plasmid.

 

The second method used was the polymerase chain reaction (PCA). It is a procedure used to amplify a small piece of DNA using polymerase. The yeast gene GPDI was the DNA strand to be amplified for the purpose of this laboratory practical.

 

The DNA fragments resulting from the utilization of the two methods were analyzed using gel electrophoresis. The bands were then visualized using the staining solution ethidium bromide.

 

 



Results

 

I)                   The yeast genomic DNA was prepared using the following procedure:

a.       15ml of YPD were inoculated and grown overnight at 30° C

b.      the tube containing the overnight culture wasw centrifuged at 300rpm for 5min

c.       the supernatant was removed and the cells were resuspended in .5ml of water , transferred in a 1.5ml tube and centrifuged at 13000rpm for 30s

d.      cells resuspended in 200ul breaking buffer, 200ul of .5mm glass beads and 200ul of PCI

e.       tubes vortexed for 3 min

f.        200ul of TE added and vortexed briefly

g.       then the tubes were centrifuged for 5min at 13000rpm

h.       the aqueous layer was transferred to a clean tube and 1ml or EtOH was added and mixed by inversion

i.         the mixture was centrifuged at 13000rpm for 10min and kept on ice

j.        the resulting pellet was dried out and 400ul of TE and 2,5ul of RNase A (10ul/ul) was added

k.      then the solution is incubated for 15min at 37°C

l.         then 10ul of ammonium acetate and 1ml of EtOH were added and mixed by inversion

m.     after the solution was centrifuged for 10min at 13000rpm the pellet was dried and 100ul of TE was added

 

II)                 The restriction digestion of plasmid (pYX212-DAK1) was prepared in the following way

a.       To 3ul of plasmid were added:

                                                               i.      1ul of cutting buffer

                                                             ii.      1ul restriction nuclease KpnI

                                                            iii.      5ul H2O

b.      the solution was placed at 37°C and later refrigerated.

 

 

III)              In order to get GPDI gene amplified, PCR had to be performed. The following steps were made to prepare the PCR sample:

a.       To 3ul of genomic yeast DNA were added:

                                                               i.      72ul of H2O, 1ul of P1 and 1ul of P2 primers, 4ul of dNTP, 10ul of PCR buffer and 0,8ul of Taq-polymerase

b.      the prepared solution was placed in thermal cycle block for 30 cycles using melting temperature of 94°C and annealing temperature of 43°C.

 

 

IV)              The samples from yeast and plasmid had to be separated using gel electrophoresis. Firstly, the gel had to be prepared and then the samples loaded into the wells.  The gel was prepared in the following way:

a.       1% agarose solutin – 0,6g of agarose was dissolved in 50ml of 0,5xTAE and the solution was heated until completely dissolved. 2,5ul of EtBr was added

b.      the agarose solution was poured into a casting chamber and a comb was introduced to form wells



c.       the samples were then loaded into the buffer with loading due (LD) in the following sequence:

                                                               i.      size marker

                                                             ii.      genomic DNA

                                                            iii.      PCR product (yeast GPDI gene amplified)

                                                           iv.      Uncut plasmid

                                                             v.      KpnI digest of plasmid

 

 

V)                The gel electrophoresis was run at 100V for 45min until the blue front reached 2/3 of the gel length

 

 

VI)              The gel was then observed under UV light to distinguish the different banding patters of the separate DNA products.

 

 

VII)           A calibration curve was made using GeneRuler DNA Ladder.1 A larger scan of the ruler was printed out, the migration distances (in mm) were measured and plotted against the corresponding molecular weight in base pairs (bp). This step was performed in order to achieve better accuracy in the calibration equation.

 

 

GeneRuler 1kb DNA Ladder                                GeneRuler 1kb DNA Laddersm0311sk.gif (755 bytes)

Figure 1 1.0% agarose 0.5µg/lane, 8cm length gel, 1X TAE, 17V/cm

 

 



Figure 2 Calibration curve obtained using GeneRuler (larger size printout)

 

This calibration curve had to be then modified in order to be useful for the particular gel. This was possible due to the linear correspondence between same size markers. Three representative points were chosen from the size marker on the gel and the following graph and equation was attained. A regression was performed yielding the equation needed for transforming our calibration curve into a useful one.

 

Figure 3 Graph for regression.

The calibration curve for this specific assay was then modified using the linear dependence equation obtained and the new curve and in order to obtain a simple linear equation it was plotted using natural log of the sizes (in bp).



 

Figure 4 Modified Calibration Curve that was used further in calculations.

 

VIII)         Using the obtained Calibration curve and calibration formula, we then calculate the sizes of the bands from the distance traveled on the gel.

 

Distance Traveled

(mm)

Equation Output

=ln(size)

Size

in Base Pairs (bp)

Uncut Plasmid

9,5

9,2104

10000,6

12,5

8,656

5744,511

Cut Plasmid

17,7

7,69504

2197,422

*16,5

7,9168

2742,979

11,4

8,85928

7039,413

**12,5

8,656

5744,511

Genomic DNA

10

9,118

9117,947

Table 1 Size of DNA bands obtained from gel electrophoresis.

 


Figure 5 Sizes for plasmid bands extrapolated from calibration curve.

 

 

Discussion

 

              I.      The expected size of the PCR product was 1176 bases which can be inferred from its location in ChrIV, from position 411822 to 412997.2

 

           II.      The expected size of the fragments from the restriction nuclease digest of the pYX212-DAK1 was:

 

a.       669 (positions 468 to 1155)

b.      7191 (positions 1155 to 8346)

c.       2170 (positions 8346 to 0 and back to 468)

The results obtained from the calibration curve for two of the bands for the cut plasmid lane are 2197,422 and 7039,413, which are very close to b. and c. The band corresponding to a. is not visible on the gel picture due to the poor resolution and the low brightness. The other two values obtained from the sample *5744,511 and *2742,979 are related to some side effects of the DNA characterization process. Both these are related to the incomplete digestion of the plasmid and accumulation of partially cut product.

 

         III.      The uncut plasmid results we obtained from the gel are 10000,6 and 5744,511. The actual size of the plasmid is 10030bp which explains the first value. The second band of size 5744,511 can be explained by the properties of the plasmid. The plasmid is a long circular structure and therefore it can travel in different conformations in the gel. The band corresponding to 5744,511 therefore is the same sequence that traveled in a different conformation.

 

        IV.      The genomic DNA value is 9117,947; there is also a band in this sample that stayed right next the well. The first value can be explained by the presence of a plasmid in the yeast well (e.g. 2u circle).3 Another possible explanation is that the highly sensitive DNA strand was broken during preparation. In the latter explanation, it would be in difficult to clarify why the DNA was cut in only one piece and no other fragments exist. The band adjacent to the well could be easily justified by large size of the uncut genomic DNA.

 

           V.      Restriction enzymes are enzymes produced by bacteria to prevent the invasion of foreign DNA, e.g. viral DNA.4 They cleave the phosphodiester bonds of DNA backbone. Restriction enzymes recognize and cut at specific places along the DNA molecule called restriction sites. Usually, a restriction site is a 4- or 6-base-pair sequence that is a palindrome. Restriction sites in plasmids are used for cloning. Restriction enzymes are used to cut the plasmid at a specific location where the gene of interest is inserted and attached using DNA lygase. The plasmid is thereafter reintroduced into an organism where it functions in the same way as it would normally. Using this method, scientist can study the function of a gene, its expression, its product, etc.



Reference

 

1 http://www.fermentas.com/profiles/electrophoresis/psm0311.htm

2 http://db.yeastgenome.org/cgi-bin/locus.pl?locus=YDL022W

3 http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/yeast-plasmid.html

4 DNA Scissors: Introduction to Restriction Enzymes.” kvhs.nbed.nb.ca/gallant/biology/restriction.pdf