Authentication of Coffee using PCR with the

E.coli Psul (BstYI) Restriction Enzyme, for

RFLP Gel Electrophoresis.

Chelsea Ann Stocks

P15228982
May 2018

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

Contents
1 Introduction 3
1.1 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Predictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 Literature review . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2 Method 8
2.1 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4 Psul . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.5 Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3 Results 12
3.1 Gel 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2 Gel 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3 Gel 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4 Discussion 18
4.1 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.2 Method analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.2.1 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.2.2 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.3 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.4 Psul . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.2.5 Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . 22
4.3 Additional points . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5 Conclusion 23

6 References 23

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

1 Introduction

1.1 Acknowledgements

I would like to thank my supervisor, Emma, for all the support and guidance
that she has provided throughout my project and my degree. I would also like
to thank all my other lecturers for these past three years, especially Richard
and Mark for being so understanding about personal issues.

I would also like to thank my family and friends for encouraging me to carry
on, I still struggle through everyday but I can look back at this and know I did
it, and did not let anything beat me down for long.

1.2 Abstract

Two coffee plants, Sudamerika and Robusta, have undergone authentication

methods to investigate the possibility of distinguishing the two coffee strains
using their DNA. Coffee authentication is a very large topic, there are many
different strains of coffee and to tell them apart can be very difficult. This
topic needed to be investigated due to the large numbers of counterfeit coffee
in an attempt to regulate higher quality coffee. Coffee beans were roasted for
different periods of time and ground, raw coffee beans were also ground. Then
DNA extraction (Plant column extraction) was performed on 0.2g. Column
extraction is the process of breaking a cell down to DNA and debris, then
binding the DNA to a silica membrane and separating it from the debris. This
is an effective method because everything that is not DNA will be washed away.
PCR (Polymerase chain reaction) was then performed at conditions specified
later, this involves amplifying small amounts of DNA into a large number of
copies by using thermal cycling. This is the process of heating and cooling the
DNA to disrupt the bonds between strands so duplicate strands can join to the
complimentary base pairs. Restriction enzymes were then used to cleave at the
restriction site to allow for RFLPs (Restriction fragment length polymorphisms)
to form. This was then run on a gel for 30 minutes and the results were recorded.
The results were not what was hoped for, but left some room for discussion and
interpretation.

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1.3 Hypothesis

(1) Psul (BstYI) can be used to distinguish between Sudamerika and Robusta
coffee.

(2) Psul (BstYI) cannot be used to distinguish between Sudamerika and Robusta
coffee.

(3) It is inconclusive as to whether Psul (BstYI) can be used to distinguish

between Sudamerika and Robusta coffee.

1.4 Predictions

Due to research, the prediction is that the restriction enzyme will cleave one
or both of the coffee strains and produce two bands during electrophoresis to
identify the individual types of coffee. The literature in this study explains how
the different strains of coffee are identified using RFLPs.

1.5 Literature review

This section reviews literature for the purpose of weighing up different tech-
niques and methods for this experiment. The literature that is analysed will be
named as “Lit 1”, “Lit 2”, etc. A full list will be provided in the reference
section. Some techniques reviewed are for the purposes of understanding and/or
further research. In the discussion this section will be referred to, to compare
results of this study and those like it.

“Freeze-drying” is a suggested dehydration technique for grinding coffee beans

before extraction – be it roasted or unroasted. Lit 1 (Mellor & Bell 2003)
explains the “basic process” of freeze-drying and it’s effect on flavour. Though
this doesn’t mention any effect to the DNA, if it lowers the quality and the
effects DNA extraction, amplification or cleaving (use of Restriction enzymes to
produce Restriction Fragment Length Polymorphisms – RFLP’s) of said DNA.
This is not specific to Coffee instead it’s a general Freeze-Drying explanation.
Freeze-Drying is also known as Lyophilisation, discarding the water as vapour,
the rate of which relies on heat and/or vapour pressure. The article explains

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the economic benefits to the process for items such as foods and medicines.

Lit 2 (Clarke 2003) explains the specific composition of coffee beans during
freeze-drying and the process of making instant coffee. In Lit 2 it is also ex-
plained that instant coffee gives a higher yield of solubility compared to the
“house-hold brews” – with additional carbohydrates being extracted from the
bean due to the dry-freezing. In this study the difference between the DNA of
two coffee strains is to help with coffee authentication though if the make up of
instant coffee could potentially be different, a different study into instant coffee
comparison would be needed as this study uses “green” coffee beans. This study
is useful in understanding how instant coffee differs from home roasted coffee,
and suggests that instant coffee is more water soluble which can mean easier
DNA extraction. Though roasting coffee degrades that DNA.

Lit 3 (Spaniolas et al. 2006) is an article that explores the authentication of

coffee using RFLP analysis. This is very similar to this study except for the
types of coffee beans used. This study starts with DNA extraction after grinding,
using the “GeneSpin DNA extraction kit.” This is a column extraction kit. The
extracted DNA then underwent quantification. The polymerase chain reaction
(PCR) was employed alongside restriction enzymes specific to the “chloroplastic
genome.” The study goes into depth about the specific RFLP targeted. In the
study, the conclusion is drawn that the coffee beans used can be discriminated
from each other. The target region used showed the differences for Arabica and
Robusta beans used as subjects. Using this region has an advantage due to a
more sensitive detection because of the low copy number.

Lit 4 (Ferreira et al. 2016) presents a way to detect and quantify traces of barley,
corn and rice in coffee so as to establish the coffee’s authenticity. The types of
coffee under study are Arabica and Robusta. The study was performed both on
roasted and unroasted beans, and notes that during the DNA extraction there
was a lower yield of DNA for roasted beans as compared to unroasted beans due
to the high temperatures sustained by the roasted beans. The study concludes
that many cheaper brands of coffee contain traces of barley, corn or rice and
that authorities be more attentive in testing the authenticity of coffees. This
links in well with this study as part of it is comparing the electropheragram
results of roasted and unroasted beans.

Lit 5 (Desloire et al. 2006) compares four different DNA extraction techniques;
Cethyl Trimethyl Ammonium Bromide (CTAB) method, Chelex method, Qia-
gen DNA extraction kit and Filter-based technology method. These were ex-
plored using “mites”, both unfed and fed (on blood). The discussion explores
the comparison of the methods, CTAB and Qiamp (using a separation column)
gave the most efficient results with a near 100% yield! Chelex was next with

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67% as it has a ratio showing contamination. Whereas the other two obtained
a ratio showing their DNA was pure and uncontaminated. Chelex showed and
even lower yield for fed mites, suggesting the presence of blood creating a lower
yield. This could be interesting to explore the separation of blood from DNA
using different techniques. The FTA was similar to the results of CTAB and
Qiamp when tested on the unfed mites. On fed mites it held a lower yield,
though this could be improved by adding an extra purification step. As there is
no blood in this study any of the extraction techniques, barring Chelex, would
hopefully yield the same results.

Lit 6 (Spaniolas et al. 2008) is regarding the efficient and high-yield extraction
of DNA from, among others, roasted Arabica coffee beans. In discussing the
extraction of DNA from roasted coffee beans, CTAB is the extraction method
with the highest-yield. This contrasts the results for unroasted, green coffee
beans where CTAB provided the lowest yield. The quality of the DNA extracted
using the CTAB method was often too low to sustain PCR however. For that
reason, the author conclude that the GeneSpin extraction kit is potentially
the most suitable method for extracting DNA from roasted coffee beans. The
authors suggest that the GeneSpin extraction kit could be used to distinguish
between Arabica and Robusta for the purposes of authentication of coffee.

Lit 7 (Lee et al. 2012) is an article describing a protocol for the separation of
DNA fragments using agarose gel electrophoresis. The article goes into depth
about the factors in the DNA travelling along the gel, which are the size of
the DNA molecule, the concentration of agarose, the DNA conformation, the
voltage applied, presence of ethidium bromide, the type of agarose and the elec-
trophoresis buffer. Following this protocol, the DNA fragments can be visualised
with UV light, allowing for easy observation of the constituents.

Lit 8 (Tornincasa et al. 2010) describes a method of detecting the presence of

Arabica or Robusta in a sample using multiplex PCR. The method consists of
having three probes - one specific to both Arabica and Robusta, one specific
to Arabica but not present in Robusta and one present in Robusta but not
Arabica. The method outlined in this paper allows relative quantities of both
species to be determined, which would be useful in practice if the more expensive
Arabica had been diluted with Robusta. This is achieved by using an inhibitor
to exclude the Arabica and we can thereby ascertain the quantity of Robusta.
Using multiplex PCR saves time, money and effort by performing multiple PCR
reactions all at once. It also adds a more discriminatory value as there are less
restrictions on the possible reactions.

Lit 9 (Mafra et al. 2008) describes advantages of PCR-based methods as com-

pared to enzyme-based methods. Notable benefits of PCR-based methods are

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increased reliability and specificity. The sensitivity of new, PCR-based methods

is particularly appealing when dealing with low quantities such as when dealing
with processed meats. Because of increased regulatory requirements with re-
gards to food labelling, the article suggests further research into these methods
is required.

Lit 10 (Spaniolas et al. 2014) and Lit 11 (Rao 2014) describe an alternate
method of authenticating foodstuffs. The method applied in this study has
been to discriminate based on the length of RFLPs. A relatively new approach
to authenticating foodwares is called SNP (single-nucleotide polymorphism).
An SNP is a region, often between genes, where there is a significant (¿ 1%)
variability within a population. These SNPs are selected so that they show a
high inter-specific variability, but a low intra-specific variability. They are the
most common form of genetic variation within a species. Selecting multiple
regions where intra-specific variability is low, it allows discrimination based
on the presence of an SNP, as these regions are likely to be different across
different species. The choice of region is highly dependent on the subject under
scrutiny. For animals, DNA markers within the mitochondrial DNA have been
deemed adequate to discriminate between species. These regions do not show
sufficient inter-specific variability for plants. SNPs might provide a preferable
alternative to other methods based on their frequency, their discriminatory value
(which can be up to 100% for some species and regions) and the high quality
of sequence recoverability (given the right regions). There is an international
initiative called the Consortium for the Barcode of Life to developing DNA
barcoding (discriminating on the basis of SNP markers). This technique was
not applied as it was deemed too costly and time-costly.

Lit 12 (Ghovvati et al. 2009) is a study involving the identification of meat

products by using multiplex PCR. This enables the detection of different types
of meat at once, as all of the DNA samples are amplified. Matching these re-
sults from the mutliplex PCR procedure against known DNA samples of poultry,
ruminant and porcine. Lit 13 (Henegariu et al. 1997) describes a protocol for
performing multiplex PCR. As a result of using multiplex PCR, less reagent has
to be used and less time spent on developing the mixtures leading to lower costs.
Multiple primer sets are added to a single PCR mixture, whereby variable-length
DNA sequences corresponding to the primer sets are amplified. There are down-
sides to using multiplex PCR: self-inhibition whereby some primers will inhibit
the PCR process, relatively worse amplification efficiency and inconsistent re-
producibility. In this study multiplex PCR was used as a cost- and time-saving
measure, in amplifying the RFLPs of interest. Without multiplex PCR, two
separate PCR experiments would have had to have been carried out and there
were no significant downsides detected, making this a valid application.

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2 Method

2.1 Preparation

Fourteen samples were prepared, seven from each coffee type, Robusta and
Sudamerika. These were labelled R1 through R7 for Robusta and S1 through
S7 for Sudamerika. Each had different preparation but identical extraction and
analysis. S1, S2, R1 and R2 were all unroasted coffee beans grinded using liquid
nitrogen. Liquid nitrogen was added to the beans and ground using a pestle
and mortar. R3 and S3 were also unroasted but these were ground using a
coffee grinder, and no liquid nitrogen, this is to compare the effects of liquid
nitrogen on DNA extraction. R4 and S4 were roasted at 250 ◦C for two and a
half minutes before being ground using the grinder. R5 and S5 were roasted for
five minutes and then ground. R7 and S7 were roasted for ten minutes at the
same temperature before being ground using the grinder. R6 and S6 were both
roasted at the same temperature for five minutes again. The previous beans
were roasted at separate times to prevent contamination, R6 and S6 however
were roasted in the oven together to observe if this makes a difference to the
results. During the roasting the beans were agitated every minute to ensure
roasting on all sides.

2.2 Extraction

The “Isolate II, Plant DNA Kit” by Bioline was used for the extraction process.
This extraction kit included using column extraction to isolate the DNA of the
coffee beans ready for the amplification process. Around 0.2 g was used of each
sample. The weights used for each extraction are in table 1 included in the
appendix. The following steps were used and taken directly from the manual
by Bioline (Bioline n.d.), step 2.1 not 2.2 was used:

8.1 Standard protocol for purifying plant genomic DNA The ISO-
LATE II Plant DNA Kit includes two different lysis buffers for opti-
mal results with most common plant species. Please refer to section
7.3 for choosing the optimal lysis buffer system for your plant sample
and for information on how to process more sample material than
in the standard protocol. Before you start:
• Ensure Wash Buffer PAW2 and RNase A are prepared (section
7.4)

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• Preheat Elution Buffer PG to 65 ◦C

1. Homogenization
Homogenize up to 100 mg wet weight or up to 20 mg dry weight
(lyophilized) plant material. Refer to homogenization methods
(section 7.2). Proceed to cell lysis using Lysis Buffer PA1 (step
2.1) or alternatively Lysis Buffer PA2 (step 2.2).
2. Lysis
2.1. Cell lysis with Lysis Buffer PA1
Transfer resulting powder to a new tube and add 400 µL
Lysis buffer PA1. Vortex mixture thoroughly. Note: if
sample does not resuspend easily e.g. due to plant powder
absorbing too much buffer, add more Lysis Buffer PA1.
Note that the volumes of RNase A (step 2.1) and Binding
Buffer PB (step 4) have to be increased proportionally.
Add 10 µL RNase A solution and thoroughly mix sample.
Incubate at 65 ◦C for 10 min. Note: For certain plants,
increasing incubation time to 30-60 min may be required.
Proceed to step 3.
2.2. Cell lysis with Lysis Buffer PA2
Transfer resulting powder to a new tube and add 300 µL
Lysis Buffer PA2. Vortex mixture thoroughly. Note: If
sample does not resuspend easily e.g. due to plant pow-
der absorbing too much buffer, add more Lysis Buffer PA2.
The volumes of RNase A, Precipitation Buffer PL3 (step
2.2), and Binding Buffer PB (step 4) however, have to
be increased proportionally. Add 10 µL RNase A solution
and thoroughly mix sample. Incubate at 65 ◦C for 10 min.
Note: For certain plants, increasing incubation time to 30-
60 min may be required. Add 75 µL Precipitation Buffer
PL3, mix thoroughly and incubate for 5 min on ice to pre-
cipitate SDS completely. Proceed to step 3.
3. Filter crude lysate
Place an ISOLATE II Filter (violet) into a new Collection Tube
(2 mL) and load lysate onto column. Centrifuge for 2 min at
11,000 x g. Collect the clear flow-through and discard the ISO-
LATE II Filter. Repeat the centrifugation step if not all liquid
has passed through the filter. If a pellet is visible in the flow-
through, transfer the clear supernatant without disturbing the
pellet to a new 1.5 mL microcentrifuge tube (not supplied).
4. Adjust DNA binding conditions
Add 450 µL Binding Buffer PB. Mix thoroughly by pipetting
up and down 5 times or vortexing.
5. Bind DNA
Place an ISOLATE II Plant DNA Spin Column (green) into a

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

new Collection Tube (2 mL) and load sample (max. of 700 µL).
Centrifuge for 1 min at 11,000 x g and discard the flow-through.
The maximum loading capacity of the ISOLATE II Plant DNA
Spin Column is 700 µL. For higher volumes repeat the loading
and centrifugation steps.
6. Wash and dry silica membrane
• Add 400 µL Wash Buffer PAW1 to the ISOLATE II Plant
DNA Spin Column. Centrifuge for 1 min at 11,000 x g and
discard flow-through. Note: although washing with Wash
Buffer PAW1 increases purity, it can in certain cases reduce
the final yield slightly.
• Add 700 µL Wash Buffer PAW2 to the ISOLATE II Plant
DNA Spin Column. Centrifuge for 1 min at 11,000 x g and
discard flow-through.
7. Elute DNA
Place the ISOLATE II Plant DNA Spin Column into a new
1.5 mL microcentrifuge tube (not supplied). Pipette 50 µL Elu-
tion Buffer PG (65 ◦C) onto the membrane. Incubate the ISO-
LATE II Plant DNA Spin Column for 5 min at 65 ◦C. Cen-
trifuge for 1 min at 11,000 x g to elute the DNA. Repeat this
step with another 50 µL Elution Buffer PG (65 ◦C) and elute
into the same tube. Note: to achieve maximum yield or higher
concentrations refer to section 7.4 for alternative elution pro-
cedures.”

2.3 PCR

Coffea1R and Coffea1F are coffee primers used in the coffea genome. The
primers were diluted to 10 pmol per 1 µL by adding 237 µL to Coffea1R and
220 µL to Coffea1F then preparing a 1 in 4 dilution standard for each. 5 µL
of each extraction were pipetted into labelled PCR tubes. 1 µL of each primer
(Coffea 1R and 1F) were added to each PCR tube. 18 µL of distilled water
was added to each tube to bring the total volume to 25 µL. Two negative con-
trols, “NP” replaced the extracted DNA with distilled water but still contained
primers, “NW” was a PCR tube with 25 µL of distilled water with nothing else.
There were 16 PCR tubes in total that had undergone the PCR process. The
PCR conditions were 95 ◦C for 10 minutes, 35 cycles of -95 ◦C for 30s, 50 ◦C for
30s 72 ◦C for 1 minute. Then 1 cycle of 10 minutes at 72 ◦C. These PCR con-
ditions were taken from Lit 3 (Spaniolas et al. 2006). The cycle lasted roughly
two hours. The process of PCR takes place when rapid heating and cooling
disrupts the Hydrogen bonds in between DNA strands, this causes them to sep-

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arate and new ”complimentary” base pairs to attach to the ”parent” strands.
This is known as annealing.

2.4 Psul

Psul (BstYI) by Thermoscientific was the restriction enzyme that was used. This
restriction enzyme source is the E. coli gene Pseudomonas stutzeri. The protocol
that was used was digestion of PCR products directly after amplification. Ten
microliters of the PCR reaction mixture (suggested 0.1 – 0.5 µg of DNA – though
NanoDrop was not available) was added to 18 µL of distilled water, 2 µL of 10x
buffer B (standard with the kit) and 1.5 µL of Psul. This mixture was then gently
vortexed for a few seconds. Then placed in a water bath at 37 ◦C to incubate
for 1 hour and 15 minutes. For the first and second sets of results, directly after
incubation, the samples were subjected to gel electrophoresis. The third set of
results followed all of the previous steps, but then, after the incubation period,
was incubated further at 80 ◦C for 20 minutes to inactivate Psul. After Psul
was inactivated the third set of results were subjected to gel electrophoresis.

2.5 Gel electrophoresis

450 mL of distilled water were added to 50 mL of 5×TBE to make up 0.5×TBE.

100 mL of 0.5 × TBE was used to make 2% W/V Agarose solution. This was
done by adding 2 g of Agarose powder to the 100 mL 0.5 × TBE in a conical
flask and then was gently heated and agitated in a microwave until the Agarose
powder had dissolved. After heating the solution was left to cool until it was
“hand hot”. When “hand hot”, 2 µL of Syber-Safe was added to the solution.
The solution was then poured into the apparatus to form a gel (with a comb
in place to form wells). Once the gel was formed, the remaining 0.5 × TBE
solution was poured over the gel. The comb was gently pulled from the gel and
the bubbles in the wells were expelled. 10 µL of each sample were loaded into
individual wells. Once loaded, the gel was allowed to run for 30 minutes. DNA is
slightly electronegative, so as the current flows through the gel the DNA travels
to the anode (positive electrode) from the cathode (negative electrode). This is
because the positive attracts the negative on the phosphate group of the DNA.
The gel has small holes, the percentage of agarose defines how small the gaps
are. The DNA travels through the gaps until it want anymore. Smaller DNA
fragments travel further due to them incurring less resistance and fitting through
the gaps more easily. The bands will form when there are a few fragments of
the same length.

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3 Results

3.1 Gel 1

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Image 1: Gel 1 immediately after electrophoresis
Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

Image 2: Gel 1 two minutes after electrophoresis

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3.2 Gel 2

Image 3: Gel 2, 5 minutes after electrophoresis

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Image 4: Gel 2, 5 minutes after electrophoresis

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3.3 Gel 3

Image 5: Gel 3

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Image 6: Gel 3

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Image 7: Gel 3

4 Discussion

4.1 Results

Image 1 and image 2 were taken just two minutes apart. In image 1 we can
see some faint bands whereas in image 2 the bands seem to have dissipated. In

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

image 1 the clearest bands are R1, R2, S1, S2 and S3. Faint bands can be seen
from R3 through to R6 and S4 through to S6. R7 and S7 both have no bands
visible. This suggests that the DNA degraded after being roasted for longer.
This is due to the amount of heat it was exposed to. Both negative controls
also showed no band. No clear difference can be seen between the two different
coffee strands except Sudamerika has a slightly thicker band. In photo 2 the
bands are all faded but S1 and S2. This is likely because there was more DNA
present from the clearer band. The second set of results were a repeat of the
first set to determine if the bands faded due to error in the method.

Images 3 and 4 were both taken 5 minutes after the electrophoresis had finished,
as this was around the time the first set of bands dissipated. As can be seen
in both photos, there are no bands visible, so this suggested that the error was
due to the method. Upon further research, the error was discovered to be that
the restriction enzyme (Psul) was not deactivated after incubation. This was
rectified in the third experiment by incubating the samples further at a higher
temperature to deactivate the restriction enzyme.

Images 5, 6 and 7 are all of the third gel. Image 7 is the most in focus, so I
will be using that to analyse the third set of results. The samples from top to
bottom are as follows; S7, S3, S1, R7, R3, R1, NP and NW. I selected these
six samples as I was limited for the restriction enzyme, so I chose the six that
I thought would best represent the result set. The negative controls were both
negative, which shows no primer dimer and no contamination. It can clearly
be seen that there are bands for R1, R3, S1 and S3, whereas the bands for S7
and R7 seem to be less apparent. This could be due to the first four samples
having been unroasted. The addition of roasting leads to degraded DNA which
understandably reduces the amount of DNA shown in the bands. In this photo,
like in 1, the Sudamerika bands are thicker than the Robusta, though there are
not individualising bands with this restriction enzyme.

4.2 Method analysis

4.2.1 Preparation

Liquid Nitrogen was added directly to R1, R2, S1 and S2. Lit 1 (Mellor & Bell
2003) includes a description of preparations that make the freezing more suc-
cessful. When in the lab, after adding liquid nitrogen, the beans were ground. In
the lab the beans were only frozen, not Freeze-dried. This could have improved
DNA extraction, equally it could have degraded the DNA due to the addition

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of heat. Additional experiments could investigate this. Lit 2 (Clarke 2003)

added that freeze-drying to prepare instant coffee can add soluble substances to
the coffee, an investigation into the effects this would have on the extractions
and analysis would be helpful for a study on the dry-freezing effects. Another
point to make would be that the beans were ground in the same coffee grinder
– cleaned thoroughly between each sample, but this could still have caused con-
tamination. The beans were roasted in a home-oven, which again could produce
the possibility of contamination (from inside oven, utensils, trays, etc.) or in-
accurate timings or temperature. A downside of freeze-drying is the additional
time it takes. Lit 1 explains each tissue type for freeze drying can take house
(around 8-16) to complete. The plant lab have access to a mechanical grinder
that helps with grinding after the use of liquid nitrogen, this would be useful
in further investigation. Another possibility would be to bath the beans in wa-
ter and other liquid for a long period of time before adding liquid nitrogen, to
observe the effects of this.

4.2.2 Extraction

The extraction method and kit was followed and used correctly, column ex-
traction is a relatively effective technique at separating the desired DNA from
the unwanted debris. Unfortunately, DNA that is similar (I.E. different strands
of coffee DNA) would not be separated from one another. If the coffee beans
had been contaminated with each other, the extraction technique wouldn’t have
helped. Quantification of the DNA would have also been desirable as it would
show the effectiveness of the extraction technique and the difference in DNA
yield from roasted and unroasted samples. Due to the unavailability of the
NanoDrop and time constraints, this was not able to be completed. Qubit
could have also been used. It is clear from the results the extraction were suc-
cessful due to the band appearing though it doesn’t rule out the possibility of
contamination from the other beans.

4.2.3 PCR

The polymerase chain reaction amplifies DNA. Due to the unavailability of

quantification the PCR efficiency couldn’t be calculated, though this would be
a point for further investigation. In Lit 3 (Spaniolas et al. 2006) They quantify
and isolate the pure amplicons for a greater DNA density, for clearer bands.
Multiplex PCR can be used as a more complex version of this study. This
would benefit coffee authentication greatly if the right multiplexing sequence
was included, multiplex is good because it takes multiple STR (Short Tandum

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

Repeats) over multiple loci, the more STR’s used the more discriminatory due to
more specific combinations. This would be great for coffee considering there are
over 100 coffee strains, but since Arabica and Robusta are the most commonly
cultivated (Alongside sudamerika) you would have to weigh up and think realis-
tically what they are more likely to use for a counterfeit. They would probably
swap for a cheap alternative, but a commonly used one, or try to add things,
like carbohydrates.

4.2.4 Psul

The restriction enzyme in this study was BstYI from the Psul gene (based in
E.coli). Below is an image showing the restriction site in the DNA:

Image 8: Psul restriction site

The bands of the DNA are thicker for Sudamerika, this indicates that the di-
gestion hadn’t fully occurred as there were not two separate band as would be
expected. The results of the digestions could be due to a multitude of reasons.
The suggested time for digestion was 1-16 hours. Perhaps a longer time would
have facilitated a completed digestion. Another reason could be that due to
the DNA not being quantified the ratio of DNA to restriction enzyme could
have been too high, therefore not all the DNA would be completely digested.
Another possibility could be contamination. If Robusta had been in the Su-
damerika sample then that would explain why they had not separated as the
Robusta would not digest. In the other studies the two coffee strains were Ara-
bica and Robusta. These two are not the same and therefore the restriction
enzyme could be wrong for the targeted site. The site targeted is the region
shown above in the chloroplastic region. The primers may also fall into this
category as they could be better used for Arabica and not Sudamerika.

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

4.2.5 Gel electrophoresis

If the digestion had been complete then it would stand to reason that the bands
didn’t separate due to the gel either being too dense or not dense enough.
This could be tested by using different W/V for the agarose Gel, or perhaps
running the gel longer to give the DNA more time to separate. Some types of
electrophoresis also quantify the samples, that would be a good further research
tool. It would also be great to identify the quality of the DNA through repeated
testing.

4.3 Additional points

Different restriction sites could be used to allow for a more discriminatory profile.
Fluorescent markers could also be used, though this may outweigh the usefulness
due to the expense. It is clear that longer roasting times degrade the DNA
samples. A further point of research could be to investigate the effect of different
temperatures at the same time and see the degradation rate. You can do the
same with the coffee beans with the quantification at different times and create
some graphs to determine at which point the DNA is so degraded that there is
no way to tell them apart.

The possibility of contamination must be discussed. Contamination can occur

throughout the experiment. A few examples would be; coffee beans being mixed
up or mixed together, the coffee grinder being washed poorly, pipette tips being
changed infrequently or not at all, cracks in the wells of the electrophoresis
gel. There was no contamination in the controls, this implies if there was any
contamination it would have occurred before the control samples were made,
which is before or during extraction. The study could have benefited from
more duplicate results. Due to time constraints and less Psul than originally
thought this was unable to happen. Extra techniques would have also been
beneficial and are a good point for further research, such as quantification,
trying different incubation periods to compare. More coffee plant types would
make for an interesting paper too. Due to this being an authentication analysis,
trying different combinations of primers and restriction enzymes and comparing
the results would make a great data set.

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Chelsea Ann Stocks P15228982 Forensic Science BSc(Hons)

5 Conclusion

Robusta and Arabica coffees can be identified from each other using chloroplastic
restriction enzymes, but it is uncertain whether Robusta and Sudamerika can
be. It is not certain if the restriction enzyme is an individualising restriction site
between the two sets of DNA. Hypothesis three seems to have been the outcome
which contradicts the predictions. With more time and further research the
questions and additional points could be answered and so could the aim of this
project. This thesis explored the possibilities to make the best of interpreting
the results obtained. Though not conclusive the study has encouraged lots of
interest around the subject and possibility of further study/research. The results
and research indicated that it should be possible to extract the DNA and identify
the two strains genetically. Perhaps this was not the right restriction enzyme
to help differentiate between the two, or perhaps it wasn’t left long enough to
incubate. The results are inconclusive and open to interpretation, the discussion
explores more ideas for further study.

6 References

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Clarke, R. (2003), {COFFEE} — instant, in B. Caballero, ed., ‘Encyclopedia of
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Desloire, S., Moro, C., Chauve, C. & Zenner, L. (2006), ‘Comparison of four
methods of extracting dna from d. gallinae (acari: Dermanyssidae)’, 37, 725–
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Ferreira, T., Farah, A., Oliveira, T. C., Lima, I. S., Vitório, F. & Oliveira,
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Henegariu, O., Heerema, N., Dlouhy, S., Vance, G. & Vogt, P. (1997), ‘Multiplex
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Mafra, I., Ferreira, I. M. & Oliveira, M. B. P. (2008), ‘Food authentication by

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Appendix

Sample - Robusta Weight (g) Sample - Sudamerika Weight (g)

R1 0.2033 S1 0.2067
R2 0.2087 S2 0.2149
R3 0.2059 S3 0.2084
R4 0.2098 S4 0.2108
R5 0.2096 S5 0.2018
R6 0.2125 S6 0.2068
R7 0.2041 S7 0.2123

Table 1: Weights of powdered sample used for extraction

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