BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY/

MEDICAL LABORATORY SCIENCE

Molecular Biology and Diagnostics

Written Report - Journal on PCR Variant

Strand Displacement Amplification (SDA) Journal Review:

“Trigging Stepwise Strand Displacement Amplification
Lights Up Numerous G-quadruplex for Colorimetric
Signaling of Serum MicroRNA”

Presented by:
Castillones, Kyla Marie S.
Cruz, Marie Johanna G.
De Leon, Louisa Ally A.
Edillor, Shania Kaye A.
Enriquez, Francesca Tiffany DC.
Gabriel, Andrea Joyce P.
Garcia, Marc Angelo G.
2MT - K

Presented to:
Prof. Adlene Anne G. Atienza, RMT
Prof. Gianina Dacuya, RMT
Prof. Franchesca Marie Lacuesta, RMT

March 12, 2023

 STANDARD DISPLACEMENT AMPLIFICATION (SDA)

Over the past 2 decades, SDA has been widely used as an alternative to PCR for
the detection of pathogens, hereditary diseases, and cancers. Also, nucleic acids that
have been amplified by SDA can be used in multiple ways and easily provide optical
and visual readouts. Strand displacement amplification (SDA) was first described in
1992 by Walker and coworkers.

According to the reference book on Diagnostic Molecular Biology, Strand

displacement amplification (SDA) uses the activity of a restriction endonuclease and an
exonuclease-deficient strand displacing DNA polymerase to generate copies of a target
DNA sequence (Shen, 2019). Furthermore, SDA is carried out under isothermal
conditions. It is based on how RNA transcription and DNA replication happen in the
body normally, which happens at constant temperature.

According to Kamiya 2022, Strand displacement amplification (SDA) is a

molecular technique used to amplify nucleic acids, like deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA). It is a common technique in molecular biology and an alternative
amplification method to a polymerase chain reaction (PCR). Unlike PCR, however, SDA
is an isothermal process, which means the procedure takes place at a constant
temperature and does not require changes in temperature like PCR.

Strand displacement amplification is another variation of the rolling circle theme

but differs from rolling circle amplification (RCA) in that the amplicons are displaced
from a linear template and do not generate concatamers (Walker et al., 1992a). The
SDA reaction occurs in two stages: (i) duplication of the target sequence by DNA
polymerase resulting in the addition of restriction endonuclease sites at each end of the
amplified target and (ii) exponential amplification consisting of multiple rounds of
restriction endonuclease nicking, an extension of the nick by DNA polymerase, and
strand displacement (Walker et al., 1992a).
Requirements of Strand Displacement Amplification (SDA)
2 primers:
● Bumper primers
○ Primers on the outside.
○ Designed as standard PCR primers
○ Displacing newly created strands.
○ DNA polymerase makes both sequences at the same time, and the
bumper sequence moves the target sequence.

● SDA primers
○ Primers on the inside bind to the restriction site and make it easier to
multiply the target sequence.
○ Bound just next to the bumper primers at the target sequence. This
method comes with a number of applications
○ Copying the base sequence
○ Contains a special restriction enzyme cut site and is specific for a known
region of the template DNA
○ Always binds just upstream of where the SDA primers bind.

Process of Strand Displacement Amplification (SDA)

1. Target Generation
- In the first step, it is called "target generation”, and involves modification of the
target sequence. The goal of the target generation phase is to generate a new
special version of the DNA target we want to amplify.
- The heat breaks down double-stranded DNA into two single stranded copies.
- Specially manufactured primers combine with DNA polymerase to form altered
targets capable of exponential amplification.
- The inner and outer primers are made to attach to certain parts of the target.
Process of Target generation:
A. DNA are separated

B. Comes the two primers

C. Position the SDA primer binds

D. Bumper primers binds upstream of the SDA primers

 E. DNA polymerase binds to both SDA and bumper (both primers are
extended at the same time)

F. SDA primer, which is now fully extended, leaves its template DNA

2. Exponential Target Amplification

- During the second stage of SDA, the nucleic acid made by DNA polymerase from
the SDA primer is made bigger. The nucleic acid, on the other hand, is not the
same as the original template of DNA (or RNA). It has been changed so that
nicking enzymes can recognize it.
- So the strand which was left or displaced in the previous step will act as the
template during amplification phase of the target
Process of exponential target amplification:
A. Modified strand is now hybridized to its complementary strand.

B. Specific restriction site is recognized by an exonuclease

C. the axon nucleus will only cut (nick) at a particular single end

D. DNA polymerase is tasked for identification and recognition of this nick

site.
 E. The polymerase starts making new strands of DNA.

F. Action of polymerase moves the previous pattern. The process of nicking,

moving, and nicking again happens over and over.

Relevance of Standard Displacement Amplification (SDA)

According to Anne Kamiya (2023), The molecular technique known as "strand

displacement amplification" (SDA) is used to amplify nucleic acids like DNA and RNA. It
can be used as an alternative to a polymerase chain reaction (PCR), it is a common
method in molecular biology. SDA, on the other hand, is an isothermal process, unlike
PCR, which necessitates temperature changes but is carried out at a constant
temperature.

In the medical field, specifically for medical technologists, this kind of method has
the potential to significantly influence the diagnosis and management of a variety of
infectious diseases. It gives an impact on the early diagnosis of a disease since it
increases the chances of successful treatment, and genomics can detect disease long
before symptoms present themselves.
 It can be done in swab (Endocervical or urethral) from a patient noninvasively on
a urine sample
Some of its real life applications include the detection of:
● Pathogens
● Hereditary diseases
● HIV viral load
- It can also be used for the detection of bacteria such as:
● Mycobacterium tuberculosis
● Chlamydia trachomatis
● Neisseria gonorrhoeae
● Various cancers

Additionally;
● Strand displacement amplification (SDA) is an isothermal in vitro method
for amplifying a DNA sequence before it is detected.
● In SDA product analysis, detecting the amplified product is important for
the isothermal amplification of nucleic acids. This includes figuring out how
much of the amplified product was made in the end and keeping an eye
on the product as it was being made.
● Thermophilic SDA that is made at a higher temperature makes a cleaner,
faster product with a higher amplification efficiency.
 REVIEW OF THE SDA JOURNAL PROPER

1. What was the paper all about?

MicroRNAs, a group of endogenous non-coding RNAs, play a significant role in a

variety of biological functions, including cell proliferation, expression, and differentiation.
Abnormal expression of this is said to be related to different human genetic diseases
like cancer, cardiovascular diseases (CVD), diabetes, and neurodegenerative diseases.
Hence, quantitative detection and analysis of miRNA expression are crucial for the
diagnosis and treatment of several diseases associated with miRNA.

In this study, let-7a microRNA was detected through the use of a

Stepwise-Strand Displacement Amplification or S-SDA-based colorimetric sensing
platform. The system used consists of two linear DNA template probes, LDTP1 and
LDTP2, DNA polymerase, and nicking endonuclease or the Nt.BbVCI. LDTP1 is used to
detect a specific DNA sequence (5’-CCTCAGC-3’ or 3’-GGAGTCG-5’) and specifically
cleave the upper part of the double-stranded DNA. The target RNA will bind to LDTP1
to form a duplex conformation and this will then be cleaved by the Nt.BbVCI. Lastly, the
LDTP2 initiates the second extension and cleavage cycle. One target miRNA could be
amplified using SDA1 and SDA2's high amplification efficiency for high-sensitivity
colorimetric detection.

Furthermore, the study used different miRNAs, such as let-7a, miRNA-31,

miRNA-199, miRNA-203, miRNA-141, miRNA-26a, miRNA-145, and other random
miRNAs, to determine the selectivity of the S-SDA that is being developed by the
researchers. To show the practical application of the method that is being developed,
serum samples were collected from colon cancer patients and healthy individuals in
order to differentiate their let-7a miRNA. The results revealed that the researchers'
method was highly sensitive and precise in its ability to differentiate target miRNAs from
their mutations. Also, by looking for let-7a miRNAs in their serum, healthy individuals
were distinguished from colon cancer patients.
2. What were the objectives of the paper?

The objectives of the study are the following:

1. Develop a colorimetric sensing platform through the use of S-SDA for

detecting let-7a miRNA.

2. Increase the sensitivity by using the S-SDA while incorporating the

G-quadruplex DNAzyme-based sensing systems.

3. Determine if S-SDA is a good method for selective detection of let-7a

miRNA.

4. Distinguish between healthy people and colon cancer patients through the
detection of let7-a miRNAs in serum.

3. How is the paper significant in the contribution of knowledge in society as a

whole?

Strand Displacement Amplification (SDA) is a preferred detection tool due to its

simple operation, isothermal reaction, rapid amplification, and high sensitivity.
Nevertheless, the following emphasizes the significance of the paper to the
advancement of knowledge in society as a whole:

To cancer patients. This research is dedicated to those people who suffer from
life-threatening conditions. With Stepwise-strand displacement (S-SDA)-based
colorimetric sensing, early detection of pancreatic or colon cancer from a healthy
individual was made possible by the utilization of hemin, HEPES buffer, TMB, and
hydrogen peroxide due to its high sensitivity and selectivity.

To health professionals. This study is intended for medical scientists with a

desire to improve the accuracy and reliability of their findings. Due to the formation of
hemin/G-quadruplex DNAzyme, which was used in Stepwise-strand displacement
(S-SDA)-based colorimetric sensing, the sensitivity of Strand displacement amplification
was increased, making it superior to or on par with many other enzyme-assisted
methods.

To future researchers. This research was created to assist individuals who wish
to broaden the Strand Displacement Amplification (SDA) method. The colorimetric
sensing based on Stepwise-strand displacement (S-SDA) will serve as a guide for a
systematic investigation of possible findings. Besides that, this study can be utilized as
a source of research results for papers using Stepwise-Strand Displacement (S-SDA) to
detect cancer or other diseases.

4. Were the objectives met?

The researchers were able to accomplish the objectives since a colorimetric

sensing platform based on S-SDA has been successfully created for the detection of
let-7a miRNA. Also, they were able to show that the S-SDA method had excellent
sensitivity and selectivity for the detection of let-7a miRNA in samples with complex
structures since, in contrast to other miRNAs, a notably distinct color change and
absorbance signal was seen for this miRNA. Lastly, the researchers were able to
accurately identify healthy individuals from colon cancer patients as the let-7a miRNA in
cancer patients was much higher compared to the healthy individuals.
5. What were the methodologies employed by the author/s to achieve their
objectives?

The UV-vis absorbance measurements were performed on a Specord 210 plus

UV-vis spectrophotometer at room temperature. The G-quadruplex sequence was
nearly impossible to construct and the absorption peak was extremely low in the
absence of a target miRNA, showing that no S-SDA reaction occurs and no DNAZyme
is generated. While let-7a miRNA was present, there was a noticeable increase in
absorption, however, it was slightly less than when the G-quadruplex sequence was
used alone. The findings showed that the addition of the target miRNA led to S-SDA
reactions and the production of some hemin or G-quadruplex DNAzymes.
 Comparing let-7a miRNA to other miRNAs, we also notice a strikingly distinct color
change. The nonspecific miRNAs of the other members of the let-7 family (let-7b, let-7c,
let-7d, and let-7i) were examined at the same concentration in order to further
investigate the specificity of the S-SDA-bases approach. The miRNA family members
with one, two, or four mismatched nucleotides displayed almost minor color changes or
absorbance signals when compared to the let-7a miRNA, as shown in Figure S2. These
findings demonstrated the exceptional selectivity for let-7a miRNA relative to other
competitive miRNAs of our S-SDA-based colorimetric sensing platform.
 Let-7a miRNA was introduced to commercial fetal bovine serum (FBS) and
absorbance measurements were tracked in order to examine the viability of the
S-SDA-based colorimetric sensing technology for miRNA detection in actual biological
samples. Several amounts of let-7a miRNA were added to human serum samples
obtained from healthy people in 1000 fold and 100 dilutions to further assess the
practical applicability of the S-SDA-based sensing platform.

Serum samples from colon cancer patients and non-cancerous healthy serum
samples were taken from Jiujiang First People's Hospital, and absorbance intensity was
measured to further illustrate the practical applicability of the S-SDA-based colorimetric
sensing platform. Blood samples were centrifuged to extract the appropriate serum and
then heated at 95°C for 3 min to inactivate interfering nucleic acids-degrading enzymes
that could interfere with S-SDA detection. This simplified the detection process.

When compared to a healthy, non-cancerous sample, the sample from colon

cancer patients had much higher absorbance. According to the data, let-7a miRNAs are
overexpressed in cancer sample tissues. The method has the advantage of making it
simple for the eye to distinguish between cancer patients and healthy individuals. In
addition, miRNAs were detected blindly to show that S-SDA can differentiate between
target and nontarget miRNAs. These first findings showed that colon cancer diagnosis
utilizing clinical serum samples might be accomplished using the S-SDA-based
colorimetric sensing platform.
6. What were the conclusions drawn?

For the purpose of detecting let-7a microRNA in clinical blood samples, the
researchers developed a colorimetric sensing platform that is based on stepwise-strand
displacement amplification (S-SDA). To be able to demonstrate the superiority and
higher selectivity of the S-SDA-based method, the researchers used this technique to
detect let-7a miRNA in eleven (11) complex matrix samples that included clinical and
fetal bovine serum (FBS) samples.

Despite the fact that there were other microRNAs present in the samples, the
S-SDA-based technique generated a high selectivity toward the let-7a miRNA. With the
development of the S-SDA method, the researchers were able to come up with results
that showed high sensitivity, with a 63.2 pM detection limit and a 0.1 nM detection limit
by the naked eye. This high-sensitivity visual detection of let-7a miRNA through the
naked eye without the use of special materials or equipment was accomplished by
utilizing and taking advantage of the high amplification efficiency of a dual-stage
isothermal amplification.

Most importantly, it was made possible to identify and differentiate colon cancer
patients from healthy individuals by utilizing the appropriate identification of let-7a
miRNAs in the blood, with the help of the stepwise-strand displacement amplification,
that was developed. Moreover, the researchers believe that it is possible to utilize the
S-SDA colorimetric sensing platform’s naked eye detection in fields such as biological
monitoring and detection, and clinical diagnostics, as a result of these analytical
capabilities and features.
7. Are there any research gaps that may be addressed by future researchers?

One of the primary research limitations that can be found in this study is that it
was limited to 20 plasma samples only. On November 2022, the researchers selected
20 participants from the Jiujiang First People’s Hospital in China - 10 plasma samples
coming from colon cancer patients, and 10 plasma samples from healthy persons. The
table below shows the relevant information with regard to the sample population size.

Median Age Age Range Sex Ratio

10 Plasma 61 years old 48 - 72 7:3

Samples from
Colon Cancer
Patients

10 Plasma 60 years old 50 - 71 7:3

Samples from
Healthy Patients

Future researchers may try to increase the sample population size for more
accurate and precise data for comparison. Furthermore, they must give specific criteria
of what it means to be a “Healthy Patient” Participant. More in-depth testing of the
S-SDA colorimetric sensing platform can be done by future researchers to ensure the
accuracy of data. Moreover, the researchers have also stated that the accuracy of the
approach needs to be further verified in the case of real applications since the
interference of other substances in the blood can have a big effect on the results. The
study can provide a stronger conclusion on the efficiency of S-SDA for signal
amplification if more samples are utilized.
 The efficacy of the stepwise-strand displacement amplification (S-SDA)-based
colorimetric sensing platform on the detection of other types of miRNA, can be further
studied by future researchers. Additionally, since the study focused on colon cancer
patients, they can also try examing the plasma samples of patients with gastric tumors,
lung cancer, and Burkitt’s Lymphoma. Testing various patients with diseases concerned
with let-7a miRNA can yield extensive and comprehensive data. They can also start on
new research aiming to know if the stepwise-strand displacement amplification
(S-SDA)-based colorimetric sensing platform is capable of efficiently detecting other
forms of miRNA. These studies will be a great help and potential for clinical application
in biomedical analysis and early clinical diagnosis.

8. Personal opinions (as a group) on the paper

1. Overall, the Stepwise-strand displacement amplification (S-SSDA) - based

colorimetric sensing platform that was utilized in the study can be used for the
detection of miRNA and showed promising potential and can someday be
significant for several clinical applications.
2. In comparison with other SDA-based methods that required extensive processes
with high costs, visual sensing of miRNA becomes a rising research field that has
several opportunities for future researchers to further study on.
3. The S-SDA methodology done by the researchers showed high efficiency for
signal amplification, and it is expected that it would have more potential for
biomedical detection and clinical diagnostics if further tests are continuously
conducted.
4. Stepwise-strand displacement (S-SDA)-based colorimetric sensing is a promising
study. The study reveals that the sensitivity of strand displacement amplification
(SDA) can be increased by using different enzymes. Also, the detection of
amplified results can be used using the naked eye, which is impressive
especially since the sample used was small. This study significantly helps the
biochemical and clinical laboratories due to its huge findings.
5. Every S-SDA methodology has a drawback. The methodology used in this study
lacks accuracy because of the effect of other compounds in blood, which is why
in order to guarantee the quality and integrity of the findings, this approach still
needs to be confirmed before being used in practical applications.
6. The stepwise strand displacement (S-SDA) is still regarded as a questionable
method. As this study developed an alternative routine that has not yet been
widely investigated, applying it to laboratory routines may be a poor decision.
Researchers must investigate the method further to demonstrate its reliability.
7. The stepwise-strand displacement amplification (S-SSDA) can be applied to
rapidly detect a target nucleic acid with high sensitivity in a point-of-care setting
and has become a popular molecular diagnosis method. Also, it is helpful in
identifying critical diseases (such as cancer, cardiovascular disease, diabetes,
and neurodegenerative diseases).
8. Since traditional non-amplified methods are expensive and gives false-positive
results, new signal amplification methods have been developed such as
hybridization chain reactions (HCR), catalytic hairpin assembly(CHA), strand
displacement amplification (SDA) and rolling circle amplification (RCA).
Furthermore, these methods have helped miRNAs become easier, and an
attractive tool for the detection and analysis of miRNAs.
9. With the high sensitivity and specificity of the stepwise-strand displacement
amplification (S-SDA), and its simple and cost-effective alternative to traditional
diagnostic techniques, which often requires trained personnel and expensive
equipment and materials, this makes it a very promising diagnostic and early
detection tool for point-of-care. However, not all SDA methods are affordable and
cost-effective, as some of these require labeling of fluorescence, quantum dots,
organic dyes, and nanoparticles, which increases the cost of the process.
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