LAB CLASS:

Jesse Weber
VBSC 445
DATE:
11/11/2016
TYPING TECHNIQUE:
Randomly Amplified Polymorphic DNA (RAPD)
OVERVIEW:
Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments from PCR
amplification of random segments of genomic DNA with a single primer of arbitrary nucleotide
sequence. This is different from traditional PCR due to the obvious fact that it lacks any
requirement of specific knowledge of the target organisms DNA sequence. In addition, the
identical 10-mer primers will or will not amplify a segment of DNA, which depends on the
positions that are complementary to the primers sequence. Due to the lack of necessary
knowledge required to amplify the DNA necessary for this procedure, it makes the procedure
particularly good for organisms such as bacteria which often mutate and change at such a
marvelous rate.
RAPD utilizes the DNA observing procedure of Gel Electrophoresis. A simple procedure,
Gel Electrophoresis uses the intrinsic negative charge of DNA molecules to move them
through an agarose gel. This distance moved is based on the amount of base pairs that a
particular segment of DNA is. The longer the Base Pair, the less the DNA segment will move
when compared to a segment of smaller Base Pair number. Through using this technique, it is
often easy to determine two things: (1) How long is a particular segment DNA and often the
particular gene, and (2) how does that sample organism compare to other sample organisms.
When looking at multiple wells of the gel, it is easy to tell if organisms have the same
genes/DNA segments because they will move roughly the same distance through the gel. Of
course it is impossible to tell if they truly are the same gene without genotyping, but this is a
quick and easy way to tell if they are of the same base pair length an inexpensive and quick
way to assume they are indeed the same gene.
This is particularly good and useful in the real world due to the fact that scientists are
using DNA more than ever. In addition, this is incredibly important when determining the
Antibiotic Resistance of particular organisms. If there is a known outbreak of bacteria in an
area, and you suspect there to be a resistant population of the organism, then one can do this
particular method. By providing controls of various resistance genes and then running
unknown bacteria in the same gel, you are able to see if it has that same gene/DNA segment
if they are at the same length within the gel.
REFERENCE:
NCBI: https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/
LABORATORY EXPERIMENT
ISOLATE NUMBERS: 36 & 37
1) Source and history
Isolate 36: From Hollys Midwifery Services Outpatient. Collected 6/1/2014, the
same day that the patient was both admitted and discharged. The particular isolate
was collected via a cervical swab. The patient was a 51-year old home maker who
had an inflamed cervix, who at the time had post-menopausal syndrome
2) Source and history
Isolate 37: Collected from a female EDEN-dog aged 7 years. The particular host had
pneumonia and later passed away. The sample was collected on 5/30/2014 when the
dog was admitted to Kennel-5 at ACGS Pet Services. The sample was from nasal
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discharge.

PROCEDURE (STEPS 1-x)

1) Make 25 uL of reaction Mix
2) Calculate volume of genomic DNA template needed for final weight of 10ng.
3) Calculate the volume of water needed to bring the final reaction volume to 25 ul.
4) 25 ul reaction
10x PCR Buffer- 2 ul
10mM dNTPs- 5 ul
OPA-02 primer- 1 u
Taq polymerase- 0.25 ul
Sterile Water- x
Template DNA- y

5) Make the master mix for multiple (without template DNA) for multiple reactions to
account for pipetting errors and to maintain the consistency among reactions. Make
master mix for one extra reaction. E.g. for 3 bacterial isolates or 3 reactions, make the
master mix for 4 reactions (4 times the volume of each component excluding DNA
and water, add 8.75 ul of this master mix to 3 different tubes, then add the appropriate
calculated amount of DNA and water to each tube (due to different concentrations,
these volumes will be different in each tube. The final volume of each must be 25 ul)
6) First load the appropriate volume of master mix (without template DNA) to the labeled
PCR tubes
7) The load the appropriate volume of template DNA to each tube and close the tubes
with the lids
8) Spin the tubes to remove air bubbles and load the tubes to the PCR machine
9) The PCR cycling conditions are;
First 5 cycles:
Denaturation- 94C 1 min
Annealing- 27C 2 min
Extension- 72C 2 min
Next 45 cycles
Denaturation- 94C 1 min
Annealing- 32C 2 min
Extension- 72C 2 min
Final extension- 72C 15 min
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DAY TWO
10) Make 0.7% agarose gel
For 100 ml of gel solution
TAE buffer- 100 m.
Agarose- 0.7g
Microwave agarose/buffer until it is completely dissolved
Add 5 ul of EtBr to gel solution at 50C

11) Le Pour the gel solution and allow it to solidify

12) Load 6uL of 1kb ladder in the FIRST and LAST well of the gel
13) Mix 5 uL of loading dye with 20 uL of PCR product and then load those into the wells!
14) Run the gel at 120V for 45 min
RESULTS
Narrative:
Isolate 36:
- Band #1: 400bp length
- Band #2: 490bp length
- Band #3: 850bp length
Isolate 37:
- Band #1: 900bp length
- Band #2: 1,250bp length
- Band #3: 1,600bp length
- Band #4: 2,100bp length
- Band #5: 2,750bp length

PICTURE:

Interpretation:
Isolate 36 had a very low number of bands when compared to the other isolates. It is possible
that the specific primers did not fit this isolate as is.
Isolate 37, had a large number of bands, and in particular shared those bands with a large
number of other isolates: 16,17,18,19, 21, 22, 23, 24, 25, 27, 28, 31, 32, 33, 38, and 39. This
suggests they have similar sequences and therefore similar strain evolution.