Course name: Molecular Biology 2

Course Code: BIOL3312

Date: 31/10/2022

Title:
Identification and analysis of pTrc99A/topA by Restriction endonuclease digestion [Lab #13A
and 13B]

Aim:

1. To confirm the that the recombination between pTrc99A plasmid and the topA gene was
successful using restriction endonuclease digestion.
2. To identify the sequences within topA-cysB as well as determine its orientation within
the plasmid samples

Abstract: Summation (approximately 250 words) of background information, aim, method,

results, and conclusion. (5 marks)

Introduction:

Restriction endonucleases are enzyme that cut DNA into fragments at specific DNA sequences
called restriction sites. These restriction endonucleases are produced by bacteria as a protection
mechanism which cut invading viral DNA. When isolated from the bacteria (e.g. EcoRI from
Escherichia coli) they can be used to cut isolation DNA such as plasmid DNA at specific sites.
In this experiment, EcoRI, PvuII and BamHI were used to cut (digest) the pTrc99A/topA
recombinant DNA in order to identify and analyse the pTrc99A/topA. These restriction enzymes
cut the pTrc99A/topA recombinant plasmid as the specific restriction sites producing fragments
of varying lengths. Knowing the total plasmid size one can use the sum of the length of the
fragment in order to the determine if the pTrc99A plasmid contained the topA gene and confirm
the orientation of the top-cysB insert with the expression vector pTrc99A/topA. Using a
restriction map of a pTrc99A the known restriction sites within a sequence of DNA can be used
to calculate the expected sizes of the fragments from each restriction enzyme digestion. The
restriction endonuclease was added in excess to ensure that the plasmid DNA was completely
digested. To ensure adequate digestion, this process was conducted in a buffer with bovine
serum albumin (BSA).

In order to find the actual sizes of the fragments gel electrophoresis is used. Gel electrophoresis
separates DNA, RNA, and proteins are separated in a gel matrix based on size and charge. The
gel matrix can be made from a variety of materials, but the experiment used agarose gel
(obtained from seaweed). Agarose gel electrophoresis is a technique used to analyse DNA
molecules of differing molecular weights. It is possible to isolate nucleic acids with 300–10,000
base pairs using an agarose gel polysaccharide. The DNA sample samples are put into the wells
of the agarose gel, which is submerged in a buffer solution. The DNA fragments can fit through
the tiny holes in the agarose gel matrix, and an electric current is used to separate the fragments.
In the presence of an electric field, the negatively charged DNA fragments at the anode would
move to the positively charged electrode known as the cathode. As the pieces move through the
matrix, they separate based on molecular size (and charge), with the smaller moving fragments
passing through the matrix more quickly than the larger moving fragments. Once the gel run is
complete, the size(s) of the bands on the gel electrophoretogram can be used to determine the
overall size of the plasmid DNA using the 1kb ladder on the gel. The agarose gel is dyed with the
fluorescent dye ethidium bromide to make the separated DNA fragments visible. Ethidium
bromide is an intercalating agent that will bind to the DNA's bases and insert itself there. The
stained DNA will glow and appear as bands on the electrophoretogram when exposed to UV
light. Stronger bands would result from larger DNA fragments. The obtained bands are
compared to a typical DNA ladder in order to establish the molecular sizes. The several bands
that are present in one lane depict the various topological forms of the DNA.
The 1kb ladder on the gel and a positive control can be used to measure the size(s) of the bands
on the gel electrophoretogram after the gel run is finished to estimate the overall size of the
plasmid DNA. Using the sizes of the bands (which represent the fragments) the total plasmid size
can be calculated by summing the sizes of the fragments. It is expected that the pTrc99A/topA
recombinant DNA will be cut into eight fragments of varying lengths that add up to 7978 bp.

Method: Reference to lab manual by name including pages used. Modifications made, if any.
(5 marks)

Calculations:

 Expected sizes of the fragments from each restriction enzyme digestion.

Table 1
Restriction enzymes used to digest pTrc99A and the corresponding cut sites.

Restriction Enzyme Cut sites (bp)

EcoRI 0, 2124
BamHI 0, 2492, 3583
PvuII 0,162

Single Digest Fragment Sizes

  BamHI cut sites are 2492 bp, and 3583 bp, hence three excepted fragments

2492 – 0 = 2492bp
3583 – 2492 = 1091bp
7978 – (2492 + 1091) = 3583 bp

 EcoRI cut sites is 2124 bp hence two expected fragments

2124 – 0 = 2124
7978 – 2124 = 5854bp

 PvuII cut site is 162 bp two expected fragments

162 – 0 = 162bp

7978 – (162) = 7816 bp

Double Digest Fragment Sizes

BamHI and EcoRI cut sites are 2492 bp, 3583 bp, and 2124 bp

2124 bp – 0bp = 2124 bp

2492 bp – 2124bp = 1459 bp
3583 bp – 2492 bp = 1091 bp

7978 – (2,124 + 1459 + 1091) = 4149bp

BamHI and PvuII cut sites are 2492bp, 3583bp , 162bp

162 – 0 = 162 bp
2492 -162 = 2,330 bp
3583 – 2492 = 1091 bp

7978 – (162 + 2,330 + 1091) = 4395 bp

Results:
Fig. 1

Electrophoretogram of the pTrc99A/topA DNA digested by EcoRI, the positive control the
undigested pTrc99A/topA DNA.
Fig. 2

The 1kB DNA ladder, and the Lambda DNA/Hind III

Key

Lane Name/Symbol Band Size

A1 1Kb DNA ladder Range of sizes
A8 EcoRI Digest of
pTrc99A/topA:
7Kb
3Kb
A14 Undigested -
 pTrc99A/topA-cysB:
>10Kb

10Kb
A11 Test Control: -

7Kb

3Kb

1.75 Kb

**Band sizes are approximations.

Fig 3.
Electrophoretogram of the pTrc99A/topA DNA digested by PvuII and BamHI respectively then
double digest using: BamHI + EcoRI, BamHI + PvuII, the control for BamHI, and PvuII, 1Kb
DNA ladder, and Lambda HindIII Digest DNA Ladder.

Key
Lane Name/Symbol Band Size
A5 1Kb DNA ladder Range of sizes
A6 Lambda HindIII Digest DNA Range of sizes
Ladder
A7 BamHI Digest of
pTrc99A/topA DNA:
>10Kb

7Kb

A8 PvuII Digest of
pTrc99A/topA:
>10Kb

7.7Kb

A9 BamHI + EcoRI Digest of

pTrc99A/topA:
>10Kb

4.5Kb

White circle on 2.5Kb

Electrophoretogram
1.25Kb

A10 BamHI + PvuII Digest of

pTrc99A/topA:
>10Kb

A11 TECH Control PvuII

>10Kb

7.0Kb

A12 TECH Control BamHI +

EcoRI
>10Kb

3.5Kb
2.5Kb
1.25Kb

A13 TECH Control BamHI

 >10Kb

5.5Kb
1.25Kb
A14 Test Ctrl BamHI + PvuII
>10Kb
4Kb
2.5Kb

White square on
Electrophoretogram
1.25Kb

B) Semi log Graph

C) Table 1
Mobility in mm/cm on agarose gel electrophoresis and size of the 1 kb Ladder bands, and
expected and sizes obtained from extrapolated semi log graph of the digested pTrc99A/topA
DNA using EcoRI, BamHI, PvuII, BamHI + PvuII, BamHI + EcoRI and test control BamHI +
PvuII

Lanes Band No. Mobility on gel Excepted Size Size

(mm/cm) (bp) extrapolated
from semi log
graph (bp)
1 Kb ladder 1 3.4 10000 6400
2 3.6 8000 5600
3 3.9 6000 4600
4 4.2 5000 4000
5 4.4 4000 3600
6 4.9 3000 2800
7 5.4 2000 2200
8 6.0 1500 1550
9 6.9 1000 9500
10 8.3 500 455
EcoRI 1 3.6 2124 5600
Digest 2 4.3 5854 4400
BamHI 1 3.7 2492 5650
Digest 2 4.4 1091 3600
3 No Band 3583
Observed
PvuII 1 3.7 162 5600
2 4.3 7816 4400
3 No band Observed; only 2 fragments expected

BamHI and 1 3.7 2124 4450

EcoRI 2 4.4 1459 3200
3 5.7 1091 1750
4 7.5 4149 750
TECH CTRL 1 2.9 162 6500
BamHI and 2 4.0 2330 4000
PvuII 3 4.9 1091 2500
4 6.4 4395 1250
BamHI and 1 3.2 162 5800
PvuII 2 2.7 2330 6300
3 No band Observed; but 4 fragments were expected
4
Discussion: (25 marks)

One of the aims of this experiment was to confirm the that the recombination between pTrc99A
plasmid and the topA gene was successful; this was determined using restriction endonuclease
digestion in single and double digestions. Restriction enzymes cut DNA into fragments od f
different at specific sites called restriction sites. These fragments produced by restriction
digestion are then separated by molecular size using gel electrophoresis. Using the fragment
sizes obtained from the separation one can deduce the total size of the plasmid and/or the
recombinant plasmid which includes a specific having the known sizes of each.

In the experiment the size of the original (undigested) the recombinant plasmid pTrc99A/topA
was 7978bp. To determine if the pTrc99A plasmid contained the topA gene the recombinant
DNA plasmid was digested by EcoRI, BamHI, PvuII in single digest. The plasmid also
underwent double digested using BamHI and EcoRI as well as BamHi and PvuII. The fragments
of these digestions were then separated using agarose gel electrophoresis. Evidence of that the
restriction digestions can be observe in figures 1 and 3 were each band contained within a lane
represents a fragment of DNA. Using the restriction enzymes, it was expected that there would
be confirmation as to if the pTrc99A contained the top A gene which 3802 base pairs (bp)
however this was not achieved. The fragment sizes from the interpolation of the plots of the
expected DNA fragment sizes and the mobility of the DNA fragments using the semi-log graph
do not correspond the expected top A gene fragment size. With regards to identifying the
sequences within topA-cysB as well as determining its orientation within the plasmid samples
using restriction enzymes this aim was also not achieved. Reason for this, is the fragment sizes
obtained in the results of this experiment also did not correspond with the topA-cysB fragment
size. A possible cause for this is the incorrect placement of the topA-cysB fragment within the
plasmid. If the topA-cysB fragment was inserted backwards for example the start site and thus
cut sites (restriction sites) would be different from what is expected. As such, when digested by
the restriction enzyme the recombinant plasmid would be cut a different site for the the topA-
cysB fragment therefore the size would vary for the expectation.

Furthermore, the banding patterns observed EcoRI, BamH1, PvuII,

EcoRI/ BamHI, BamHI/PvuII digest of the recombinant plasmid in Tube 1, 2, 3 4 and 5 were as
expected for the most part. Both the EcoRI and PvuII were expected to produce two fragments,
and this was the result however, only two fragments (bands on the electrophoretogram) were
observed for BamHI but 3 fragments were expected to. Comparatively the EcoRI/ BamHI double
digest four fragments were expected and the these were accounted for on the electropherogram.
For the BamHI/PvuII double digest four fragments were expected however only two bands on
the electropherogram (two fragments) were observe red. Faint or the absence of bands on the gel
maybe due to insufficient quantity or concentration of DNA loaded on the gel.

Compare to the theoretical expected sizes calculated, what may have accounted for any
differences seen? (10)
- Limitations/ sources of error with suitable rationalization (3)
Conclusion: (5 marks)

Questions

1) By referring to the restriction enzyme mapping data in Appendix 3 (p. 198 – 214), provide a
restriction map of the recombinant plasmid, pTrc99A-topA indicating the cut sites of the
enzymes used in today’s experiment. (15 marks)

2) PvuII has two cut sites in the recombinant plasmid, one of which is located at 442 bp. Use
your gel results to estimate the second cut site for PvuII restriction enzyme and indi-cate this in
the restriction map above (5 marks).