Title: Analysis of pTrc99A/topA DNA Using Restriction Endonucleases

Aim: To identify DNA sequences characteristic of the E. coli topA and cysB genes and
confirm the orientation of the topA-cysB insert within the expression vector pTrc99A/topA using
the restriction endonucleases.

Abstract:

Four reaction mixtures were made. These reaction mixtures contained some, but
not all of the following reagents-sterile water, 10 digestion buffers, plasmid DNA (125g/ml),
EcoRI (10U/l), BamHI (10U/l) and PvuII (10l). EcoRI, BamHI and PvuII are all restriction
endonucleases and were used to digest the pTrc99A/topA-cysB recombinant at various
recognition sites on the DNA molecule. After digestion of the pTrc99A/topA-cysB recombinant
it was analyzed by agarose gel electrophoresis. The results obtained indicated that the digestions
by the restriction enzymes were successful.

Introduction:

The restriction endonucleases EcoRI, BamHI and PvuII were used to

digest the pTrc99A recombinant at various recognition sites. Enzymes work best under specific
conditions therefore a restriction buffer (NaCl, MgCl2, Tris-HCl, pH 7.6) was added to ensure
that the restriction enzymes worked properly. After digestion of the plasmid occurred, the
process of electrophoresis (agarose gel electrophoresis) was carried out. The results obtained
were analyzed and along with the restriction map drawn were used to identify DNA sequences
characteristic of the E. coli topA and cysB genes and confirm the orientation of the topA-cysB
insert within the expression vector pTrc99A/topA using the restriction endonucleases.

Method:
140-141.

As seen in Experiments in Molecular Biology: Biochemical Applications, Pages

Discussion: The primary function of restriction endonucleases is to digest DNA at their

recognition sites. This function is, however, manipulated by scientists in the lab when trying to
decipher DNA sequences of a vector or the insert within an expression vector, or to confirm the
orientation of an insert within an expression vector. In this experiment, the pTrc99A/topA
recombinant was digested with various restriction enzymes (EcoRI, BamHI and PvuII). Four
different reactions tubes were setup for the digestion of the pTrc99A/topA recombinant. Each
reaction contained one or in some cases, two of the restriction endonucleases. After digestion of
the pTrc99A/topA recombinant an agarose gel electrophoresis was done and the results analyzed.
Tube 1 contained only BamHI, tube 2 contained both BamHI and EcoRI, tube 3 contained both
BamHI and PvuII, and tube 4 contained PvuII only. The size of pTrc99A/topA recombinant is
7978 base pairs (bp). Based on the restriction map drawn for tube 1 two fragments were expected
of which the expected sizes were 1091bp and 6887bp. The fragments sizes obtained in tube 1
were 7500bp and 980bp. From this result it is evident that expected and obtained sizes after
digestion by BamHI are similar. Hence it safe to say that the digestion that took place by BamHI
was successful. Tube 2, again based on the restriction map was expected to have four fragments
of sizes 2124bp, 368bp, 1091bp and 4395bp. The experimental values obtained were 2350bp,
420bp, 1000bp and 5400bp. Again the expected values and the experiment values are of similar
size therefore, it is safe to deduce that digestion by EcoRI and BamHI was successful. Four
fragments were expected for tube 3 of sizes 560bp, 2330bp, 1091bp and 3997bp. The
experimental values obtained were 500bp, 1000bp, 2610bp and 4900bp. Again the expected and
experimental values are similar and thus the deduction that digestion was successful can be
made. Tube 4 was expected to have one fragment of size 7978bp. However, after the experiment
two fragments were obtained. These fragments, based on the restriction map drawn were
expected to have sizes of 560bp and 7418bp. The experimental values obtained for the fragments
were 560bp and 8100bp; hence digestion was successful once more. The differences that arose
between the theoretical and actual size may have been the due to the amount of time given for
the reaction to occur. If the reaction was prolonged maybe digestion would occur more
efficiently and effectively. Also, the changes in size may have to do more with the fragments
conformation rather than the reaction time. Because both the theoretical and actual value were
close to each other it means that may be, for example one fragment was in a more supercoiled or
linear state than the other.
BamHI was present in tubes 1-3; in all these three tubes there was a fragment of consistent size
of 1000bp in tubes 2 and 3, and 980bp in tube one, these fragments are consistent with the
expected fragment size of 1091bp which is respective of BamHI. Due to specific cuts being
made by other endonucleases the other expected fragment size of 6887bp for BamHI was not
consistent. One fragment size in tubes two and three was consistent with the expected fragment
size of 2404bp for EcoRI; these sizes were 2350bp and 2610bp respectively. Also, in tube 2 a
double digest using BamHI and EcoRI took place; these produced the expected number of
fragments and made almost perfect cuts based on the experimental sizes obtained. However, for

the double digest in tube 3 using BamHI and PvuII, a lot of fragments were produced although
four fragments of similar sizes to the expected were present. The reason for these differences
may be due to the fact that the insert was cut by PvuII in tube 3 where as it was in tube 2,
therefore maybe different conformations were present for both the pTrc99A vector and the topA
insert; hence giving rise to that many number of bands. In tubes 1 and 4 only one restriction
endonuclease was used, BamHI and PvuII, respectively. Whereas BamHI was expected to cut
twice and thus produce two fragments, EcoRI was expected to cut only once and thus produce
one fragment. However, on observation of the electrophoretogram two fragments were observed.
Since the one cut that PvuII makes is that of the topA insert it means that there must be a second
PvuII site present in the pTrc99A vector. This second site was deduced to be at 7860bp.
Restriction enzymes digests aid in the sequencing of unknown DNA sequence. Because the
restriction endonucleases digestions in this experiment were successful they aided in the
identification of sequences within topA-cysB and in determining its orientation in the plasmid
sample.
References:
1. Zachary F. Burton. (1997).Experiments in Molecular Biology: Biochemical Application,
Experiment 13A pages 142-144, Restriction Endonucleases pages 58-59, Restriction Map page
48, Restriction enzyme mapping data appendix 3 pages 213-214.