Course title: Biotechnology I

Course Code: BIOT3113

Date: 17/11/2022

Title
Amplification of a lacZ Gene Fragment by Polymerase Chain Reaction (Exercise 7) and
Restriction Digestion and Agarose Gel Electrophoresis (Experiment 8)

Aim

1. To amplify the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene) using Polymerase
Chain Reaction (PCR) followed by the electrophoretic separation of the digested PCR Product.

Abstract

The aim of this experiment was to amplify the lacZ gene fragment from Escherichia coli (a beta-galactosidase
gene) using Polymerase Chain Reaction (PCR) followed by the electrophoretic separation of the digested PCR
Product. Polymerase Chain Reaction (PCR) is a recombinant DNA technology method used to amplify (make
several copies of) a of lacZ gene using a thermal cycler and a variety of components. Thereafter, the PCR
product was then digested using the following restriction enzyme: using EcoRI, HindII, PstI, BamHI then
separed using agarose gel electrophoresis. To conclude, the lacZ gene fragment from Escherichia coli (a beta-
galactosidase gene) that was successfully amplified Polymerase Chain Reaction (PCR) was found to be 600bp.
The PCR product was then successfully digested using EcoRI, HindII, PstI, BamHI then electrophertically
separated. The only fragment that was matched to the expected band size of 11500bp was the one produced by
the BamHI digest. Possible explanations for why fewer observed bands were observed than anticipated include
the use of insufficient DNA template, an excessively high DNA template concentration, or an insufficient
primer concentration. Furthermore, the primers may have hybridized to a secondary site on the template or the
primer concentration may have been too high, which are two other causes for the disparity between the expected
and observed band sizes.

Introduction

Polymerase Chain Reaction (PCR) is a recombinant DNA technology method used to amplify (make several
copies of) a specific gene of interest using a thermal cycler and a variety of components. These components
include two specific primers, deoxynucleotide triphosphates (dNTP), DNA template (gene of interest), reaction
buffer, thermostable Taq DNA Polymerase (from Thermus aquaticus) and magnesium ion (Mg2+). The steps
involved in PCR include denaturation, annealing and elongation. In the first step the double stranded DNA
template is denatured using heat at 94°C for 10 minutes. Once the bonds between the double stranded DNA the
two strands separate to form two single stranded DNA. The reaction mix temperature is then decreased to 65°C
to allow the two primers to anneal to the DNA template at complementary sequences. After the primers are
annealed the Taq DNA polymerase adds dNTPs to synthesis the new strand of DNA (elongation). At the end of
the new DNA synthesis the complementary new strand is bonded to the original DNA strand is then denatured
in another cycle of the PCR. After many rounds of amplifications, the DNA template would be multiplied
several times. In this experiment, you will use the polymerase chain reaction to amplify a lacZ gene from
Escherichia coli (a beta-galactosidase gene).

Gel electrophoresis is a method used to separate DNA, RNA, and proteins in a gel matrix according to size and
charge. Although a variety of materials can be utilized to create this gel matrix, agarose gel (obtained from
seaweed) was employed in this experiment. DNA molecules with various molecular weights can be analysed
using agarose gel electrophoresis. A polysaccharide called agarose gel is used to separate nucleic acids with
300–10,000 base pairs. The wells on the agarose gel, which is immersed in a buffer solution, are filled with the
DNA sample samples. To separate the fragments an electric current is introduced, and the DNA fragments
produced by restriction digestion can pass through the minute holes in the agarose gel matrix. The negatively
charged DNA fragments at the anode would travel to the positively charged electrode known as the cathode in
the presence of an electric field. The pieces segregate according to molecular size (and charge) as they move
through the matrix, with the smaller fragments moving through the matrix faster than the larger moving
fragments. The size(s) of the bands on the gel electrophoretogram can be used to calculate the overall size of the
plasmid DNA using the 1kb ladder on the gel once the gel run is finished. To see the separated DNA fragments,
the agarose gel is dyed with the fluorescent dye ethidium bromide. An intercalating substance, ethidium
bromide will insert itself between the bases of the DNA. Under UV light, the stained DNA will glow and show
up as bands on the electrophoretogram. Larger DNA pieces would produce more pronounced bands. To
determine the molecular sizes, the resulting bands are compared to a conventional DNA ladder. The PCR
product would have been in electrophorese in an agarose gel and visualized using ethidium bromide under
ultraviolet light, and the lacZ gene.

Method

The method for experiment 7 and 8 is outlined on pages 65-74 in the Biotechnology, A laboratory Course.
(1996) textbook. Notable changes include:

1. Adjusted volumes (in experiment 7) of components used in PCR and digestion reactions as follows:

Table 1

BamHI and PstI EcoRI and HindIII

Reagent X1 (μL) X32(μL) X1(μL) X32(μL)

H20 5.5 176 6.5 208

BSA 1 32 - -
Buffer 1 32 1 32
Enzyme 0.5 16 0.5 16
DNA 2 N/A 2 N/A
Total 10 8 μL each 10 8 μL each
reaction reaction

Results
Fig. 1

Electrophoretogram of amplified the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene)
using Polymerase Chain Reaction (PCR) followed by the electrophoretic separation of the PCR Products.

100 bp DNA ladder

NEB
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A1 A12A13 A14

Lane Name/Symbol Band Size (bp)

A1 LacZ gene 600
A7 100 bp DNA ladder -
A14 pRY121 plasmid -

Fig. 2
 Electrophoretogram of amplified the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene)
using Polymerase Chain Reaction (PCR) followed by the electrophoretic separation using EcoRI, HindII, PstI,
BamHI with the 1 kilobase DNA ladder and the lambda DNA HindIII ladder

NEB 1 KB
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 DNA LADDER

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14

[Grab your reader’s attention with a great

quote from the document or use this space
to emphasize a key point. To place this text
Key
box anywhere on the page, just drag it.]
POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION
POSITION

POSITION

POSITION

POSITION

POSITION
SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14
1 Kb DNA

Lambda
Ladder

BamHI
BamHI

BamHI
HindIII

HindIII

HindIII

HindIII
Digest
EcoRI

EcoRI

EcoRI
JHCC
TI BF
KMR

PstI

PstI

PstI

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14

Fig.
3
Lambda HindIII
1 Kb DNA
Ladder

SM KO

BamHI
BamHI

BamHI
HindIII

HindIII

HindIII

Digest
DHMJ

AR BS
EcoRI

EcoRI

EcoRI
PstI

PstI

PstI

Electrophoretogram of amplified the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene)
using Polymerase Chain Reaction (PCR) followed by the electrophoretic separation using EcoRI, HindII, PstI,
BamHI with the 1 kilobase DNA ladder and the lambda DNA HindIII ladder.
 B1
A1
AKME EC SAMPLE
POSITION
PstI PstI

B2
A2
EcoRI EcoRI SAMPLE
POSITION

B3
A3
HindIII HindIII SAMPLE
POSITION

B4
A4
BamHI BamHI SAMPLE
POSITION

B5
A5
ST TC RGSO SAMPLE
POSITION
HindIII PstI

B6
A6
PstI EcoRI SAMPLE
POSITION

B7
A7
BamHI HindIII SAMPLE
POSITION
B1 B2 B3 B4 B5 B6 B7

B8
A8
EcoRI BamHI SAMPLE
POSITION

B9
A9
1 Kb DNA 1 Kb DNA SAMPLE
POSITION
Ladder Ladder
SAMPLE

B10
A10
Lambda Lambda
POSITION
HindIII HindIII
SAMPLE

B11
A11
THDB KC RD
POSITION
HindIII HindIII
SAMPLE

B12
A12
EcoRI PstI
POSITION
B8 B9 B10 B11 B12 B13 B14
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14

SAMPLE

B13
A13
BamHI BamHI
POSITION

SAMPLE

B14
A14
PstI EcoRI
POSITION

Key
 Fig. 4
Electrophoretogram of amplified
the lacZ gene fragment from
Escherichia coli (a beta-
galactosidase gene) using
Polymerase Chain Reaction (PCR)
followed by the electrophoretic
separation using EcoRI, HindII,
PstI, BamHI with the 1 kilobase
DNA ladder and the lambda DNA
HindIII ladder

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14

 B1
A1
1 Kb DNA 1 Kb DNA SAMPLE
POSITION
Ladder Ladder

B2
A2
Lambda HindIII Lambda SAMPLE
POSITION
Digest HindIII Digest

B3
A3
RC RG AA AG SAMPLE
POSITION
BamHI BamHI

B4
A4
EcoRI EcoRI SAMPLE
POSITION

B1 B2 B3 B4 B5 B6

B5
A5
HindIII HindIII SAMPLE
POSITION

B6
A6
PstI PstI SAMPLE
POSITION

B7
A7
SA DR SSSBTGKW SAMPLE
POSITION
HindIII PstI

B8
A8
EcoRI HindIII SAMPLE
POSITION

B9
A9
PstI EcoRI SAMPLE
POSITION
B7 B8 B9 B10 B11 B12 B13 B14

SAMPLE

B10
A10
BamHI BamHI
POSITION

SAMPLE

B11
A11
SS SS DM DP KW
POSITION
BamHI EcoRI
SAMPLE

B12
A12
EcoRI BamHI
POSITION

SAMPLE

B13
A13
PstI HindIII
POSITION

SAMPLE

B14
A14
HindIII PstI
POSITION

Key
Fig. 5
Electrophoretogram of amplified the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene)
using Polymerase Chain Reaction (PCR) followed by the electrophoretic separation using EcoRI, HindII, PstI,
BamHI with the 1 kilobase DNA ladder and the lambda DNA HindIII ladder with observed bands selectively
highlighted
 Key
SAMPLE

B1
POSITION

SAMPLE
POSITION

B2 B3
SAMPLE
POSITION

SAMPLE
A1 A2 A3 A4 A5 A6

POSITION

SAMPLE
POSITION

SAMPLE
POSITION

SAMPLE
POSITION

SAMPLE
POSITION
A7 A8 A9 A10 A11 A12 A13 A14

B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14

SAMPLE
POSITION

SAMPLE
POSITION

SAMPLE
POSITION

SAMPLE
POSITION

SAMPLE
POSITION

SAMPLE
POSITION
 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14

1 Kb DNA

Lambda
SM ML

Ladder
BamHI

BamHI

BamHI
HindIII

HindIII
HindIII

HindIII
TRAM
EcoRI

EcoRI

EcoRI
PstI

PstI

PstI
SG
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14

1 Kb DNA

Lambda
Ladder

DC SW
BamHI

BamHI
BamHI
HindIII

HindIII

HindIII

HindIII
CP NC
SB TC

EcoRI

EcoRI

EcoRI
PstI

PstI

PstI
Lane Name Band Size (bp)
B1 BamH1 9,000
B7 Hind III >10000,
7000,2300,1700,1200
B9 PstI 7900 3600
B8 EcoRI 3100,2900,2700

Fig. 6
Electrophoretogram of amplified the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene)
using Polymerase Chain Reaction (PCR) followed by the electrophoretic separation using EcoRI, HindII, PstI,
BamHI with the 1 DNAkilobase ladder and the lambda DNA HindIII ladder,
 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14

Key
POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION
POSITION

POSITION

POSITION

POSITION

POSITION

POSITION
SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14

1 Kb DNA

Lambda
Ladder
BamHI

BamHI

BamHI
HindIII

HindIII

HindIII
HindIII

DA KN

Digest
RA DB
SB KA

EcoRI

EcoRI

EcoRI
PstI
PstI

PstI
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14
1 Kb DNA
Negative

Negative

Negative

Negative

Lambda
Positive

Positive

Positive

Positive
Control

Control
Control

Control

Control

Control

Control
control

Ladder
BamHI

BamHI

HindIII

HindIII
HindIII

Digest
EcoRI

EcoRI
PstI

PstI
Table 2

The restriction sites for the restriction enzymes EcoRI, HindII, PstI, BamHI with calculated expected
band sizes after digesting the pRY212 and the observed band sizes from the electrophoretograms

Restriction Restriction sites Expected band sizes Observed Band

Enzyme (bp) (bp) sizes (bp)
EcoRI 2260,2880,2830,3530 2260,620,50, 650, 7920 3100,2900,2700
BamHI 11500 11500 9,000
PstI 8110, 2940,450 450,2490, 5170,3390 7900 3600
HindIII 1000,1760,6640,2300 1000,760,340,4540,4860 >10000,
7000,2300,1700,1200

Calculations

DNA concentration based on previous quantification results: 59.1 ng/ul.

  For PCR, calculations to show how that DNA should be diluted to obtain a concentration of 1µg/ml in
4µl:

Final DNA concentration = 1µg/ml

Initial DNA concentration = 59.1 ng/ul x 100

= 5910 µg/ml

Dilution = Final DNA concentration/ Initial DNA concentration

= 1/5910

Dilution Factor = 5910

Serial dilution to obtain the 1/5910:

To make a 4 ul of a 1:5910 dilution which as a dilution of 5910, a 1:10 dilution, another 1: 10 dilution and a
subsequent 1:10 dilution then a 1:5.910 can be done:

(10 x 10 x 10 x 5.910 = 5910)

For the 1:10 dilution the volume of the diluent can be 18ul while the solute would be 2ul:

Dilution = ml of sample / ml of sample + ml of dilute

= 2ul/2ul+18ul
= 2/20
= 1/10

Final volume/ Solute Volume = DF

Solute Volume = DF/Final volume

= 5.910/ 4
= 1.48ul

Transferring 1.48ul ul from the third 1/10 dilution and adding it to 4ul

4ul – 1.48ul= 2.52ul diluent to perform the 1:5910

 Sample calculation of sizes of expected fragments for at least one restriction digestions except BamHI:

PstI

Size of PstI = 11,500

 Restriction sites: 8110, 2940,450

Expect fragment (bp):450,2490, 5170,3390

450 - 0 = 450
2940 – 450 = 2490
8110 – 2940 = 5170

11,500 – 8110 = 3390

Discussion

In this experiment, polymerase chain reaction (PCR) to amplify a specific region of DNA called the lacZ gene
within the Escherichia coli (a beta-galactosidase gene). Using the electrophoretogram produced from the
electrophoresis of the PCR product (amplified lacZ gene) and size was approximately 600bp and molecular
weight was 18 ng. In actuality, the lacZ gene is about 3kb and the largest product that is seen on the gel is about
700bp. One possible reason to account for this discrepancy i.e., the band size being smaller than expected is if
the amplification was non-specific. In other words, if the primers annealed to another homologous sequence
other than the lacZ gene or non-specific binding and amplification. Limitations of polymerase chain reaction
(PCR) include the susceptibility of DNA polymerases make to errors which could result in PCR product
mutations in the PCR production. The sensitivity of PCR to contamination is high. Results that are misleading
or confusing could be caused by a trace amount of contaminated DNA.

In addition to the PCR carried out to amplify the lacz gene, electrophoretic separation of the digested PCR
products. Four restriction enzymes were used they include using EcoRI, HindII, PstI, BamHI. For EcoRI the
anticipated band (fragment) sizes (all band sizes in base pairs (bp)) were 2260,620,50, 650, 7920 however the
observed band sizes were 3100,2900,2700. Apart form obvious discrepancy between the expected and the
observed band sizes the number of fragments that were observed were less than what was expected. Moreover,
for the HindIII digest the expected band sizes were as follows: 1000,760,340,4540,4860 (all in bp) while the
observed band sizes were >10000, 7000,2300,1700,1200. This trend of a sharpe contrast was also observed in
the PstI digest where the expected band sizes were 450,2490,5170,3390 but the observed band sizes were 7900,
3600 (bands in bp). Only the fragment produced by the BamHI digest was the same as the expect band size was
11500bp. Possible reasons that less bands were observed than expect could be insufficient DNA template used
or too high of the DNA template was too high or the primer concentration was too low. Moreover, some
possible explanations of the discrepancy between the expected and the observed band sizes could be that the
primers hybridized to a secondary site on the template, or the primer concentration was too high.

Conclusion
In conclusion, the lacZ gene fragment from Escherichia coli (a beta-galactosidase gene) that was successfully
amplified Polymerase Chain Reaction (PCR) was found to be 600bp. The PCR product was then successfully
digested using EcoRI, HindII, PstI, BamHI then electrophertically separated.

Question 1

Increasing the annealing temperature as much as feasible and/or adding formamide to the reaction mixture are two more
ways to improve PCR specificity. This process typically increases the reaction's specificity, but it is ineffective when the
annealing temperatures of the two primers differ. This happens because of the annealing conditions being too lax for the
other primer while being too stringent for the primer with the lower melting temperature. The usage of primers with
variable melting temperatures can occasionally occur, even though they should ideally all have the same melting
temperature values due to experimental constraints (such as the creation of primer-dimers) and/or practical
reasons.                                      

Question 2

Magnesium must be present for thermostable DNA polymerases to use it as a cofactor during the reaction. One
of the reagents with the potential for the largest impact on the accuracy of PCR is changing the magnesium
concentration. The yield of the PCR result will typically rise as Mg2+ concentrations are increased. Increased
Mg2+ levels will, however, also reduce the DNA polymerase's specificity and fidelity.
1. By varying the annealing temperature an investigator can change the stringency of hybridization during
PCR and optimize amplification conditions for a specific primer pair and template. Temperature would
be greater than 60 degrees Celsius

2. A adjustable alteration known as "hot start PCR" considerably elongates the first denaturation time. This
adjustment can be made to cycling conditions either with or without other adjustments. Additionally, it
is frequently employed in concert with additives for the production of temperamental amplicons. In fact,
general cycling circumstances are increasingly including hot start PCR on a regular basis. It has been
shown that a hot start improves the specificity and fidelity of the reaction while also improving amplicon
yield. The goal of hot start PCR is to avoid primer dimers and non-specific priming that could happen if
the reaction is set up below the Tm.

3. Although the yield can be greatly increased by using two thermostable DNA polymerases, other factors
can have a considerable impact on the yield of longer PCR products. Since fewer partial products are
generated, higher extension durations should enhance the yield of longer PCR products.

Question 3 (page 73)                                                                                                                                                             8

marks

Prepare a reaction mixture for a restriction digest containing components from the following stock solutions: DNA (2
mg/ml), BSA (10x reaction buffer) (10X and BamHI (10 U//uI). Indicate the minimum total reaction volume required for
digesting 10ug of plasmid DNA and the order in which these reagents should be added.        

2mg/ml=   2ug/ul    

Since 10ug of DNA is being digested, 5ul of the 2ug/ul DNA is used. 

1U of enzyme digests 1ug of DNA, and 1ul contains 10 U of enzyme

10 U of enzyme will digest the 10ug DNA, so 1 ul of Enzyme is sufficient

Rxn buffer vol 10% final reaction volume 

BSA vol 10% final reaction volume

Min reaction volume 10ul

*make up to final volume with water

Question 4     
Expected sizes of the DNA fragments from the following digests of pRY121:
a. SacI

Restriction sites = 1800, 463

Expected fragments = 463bp, 1337bp, 9700bp

463 – 0 = 463bp
1800 – 463 = 1337bp
11500 – (463 + 1337) = 9700bp

b. HindlII and PstI

Restriction sites = 0, 210,1000, 8320, 11260

Expected fragment =

210 -0 = 210
1000- 210 = 790
8320 – 1000 = 7320
11260 – 8320 = 2940

11500 – (210 + 790 + 7320+ 2940)

= 240

SmaI and EcoRI

Restriction sites = 960, 5590,9120,11380

Expected fragment =

906 – 0 = 906
5590 -960 = 4630
9120 – 5590 = 3620
11380 – 9120 = 2260

11500 - (906 + 4654 + 3530 + 2260)

= 84
 c. XhoI

*Not shown on a restriction map of pRY121

Question 5 

What would be the effect on plasmid function of subcloning ("inserting") a 1.0-kb random gene fragment into:

a. The SacI site in pRY121?

If a 1.0kb random fragment were to be inserted into the Saci site of the pRY121 where the lacZ gene is the gene would be
distributed and rendered nonfunctional as the random fragment would be inserted into the protein coding sequence of the
laz gene which mean it could not be transcribed. As such, beta-galatosidase the enzyme encoded by the lacZ gene would
not be synthesized which cleaves lactose to glucose and galactose because no mRNA transcript would be syntheized,
therefor no translation would take place.

b. The SmaI site in pRY121? Nttn no coding genes there

If a 1.0kb random fragment were to be inserted into the SmaI site of the pRY121 the genes of the plasmid would remain
function as the fragment would be inserted into a non-protein coding region.

c. The PstI site in the URA3 gene in pRY121?

If a 1.0kb random fragment were to be inserted into the pstI site in the URA3 gene in pRY121 the gene function would be
distributed as the fragment would be inserted into the protein coding sequence of the gene. Orotidine 5-phosphate
decarboxylase, which is encoded by the gene URA3, is a key player in the de novo synthesis of pyrimidine
ribonucleotides. One of nature's most effective enzymes is 5-phosphate decarboxylase, which is in charge of catalyzing
the conversion of orotidine 5-phosphate (OMP) to uridylic acid (UMP). With this insertion no Orotidine 5-phosphate
would be synthesized no mRNA transcript would be syntheized, therefor no translation would occur.

References
Burton, Z. F., & Kaguni, J. M. (1997). Experiments in molecular biology: biochemical applications. San Diego:
Academic Press. (pp. 65-74)

Krentz, C. (n.d.). Polymerase chain reaction amplification of lacZ to engineer a gene sequence that can be used

to modify the size of pUC19. Ubc.Ca. Retrieved November 18, 2022, from

https://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/6/6-35.pdf

Lorenz, T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization

strategies. Journal of Visualized Experiments: JoVE, 63, e3998. https://doi.org/10.3791/3998

PCR amplification. (n.d.). Promega.com. Retrieved November 18, 2022, from

https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/pcr-amplification/

Troubleshooting PCR and RT-PCR Amplification. (n.d.). Sigmaaldrich.com. Retrieved November 18, 2022,

from https://www.sigmaaldrich.com/JM/en/technical-documents/technical-article/genomics/pcr/

troubleshooting-pcr-and-rt-pcr-amplification

Weighardt, F., Biamonti, G., & Riva, S. (1993). A simple procedure for enhancing PCR specificity. PCR

Methods and Applications, 3(1), 77–80. https://doi.org/10.1101/gr.3.1.77

What are the limitations of PCR technology? (n.d.). Aatbio.com. Retrieved November 18, 2022, from

https://www.aatbio.com/resources/faq-frequently-asked-questions/What-are-the-limitations-of-PCR-

technology