Comparison

The polymerase chain reaction

Overview

 

Definition

 

PCR is an abbreviation for Polymerase Chain Reaction (engl.).

The aim of such a polymerase chain reaction is to amplify the DNA, i.e. genetic material. This is necessary, for example, if there is not enough sample DNA available.

 

PCR simply explained

 

If the name is broken down into its constituent parts, the underlying principle of this procedure can be explained:

An enzyme called "DNA polymerase" is used within a biochemical reaction. In addition, it is a chain reaction: the end product of the reaction also serves as the starting point for a further, similar reaction.

 

The procedure of a PCR

 

How does a PCR work?

The PCR can be divided into three sections: Denaturation, primer hybridization and elongation.

PCR Diagramm Img.1.: A polymerase chain reaction is divided into three characteristic steps. During denaturation the two DNA strands are separated. This is followed by primer hybridization in which the primers bind to their respective starting matrices. Finally, the synthesis of the "new material" takes place during the elongation .

 

In the first step, denaturation, the two single strands of the double-stranded DNA are separated from each other with the help of heat. This breaks the hydrogen bonds that previously held both strands together.

For the next step, the primer hybridization, primers are necessary. Primers are oligonucleotides. They were selected to bind (hybridize) to a specific segment within the strand to be amplified.

The PCR primer design plays a special role in the course of a PCR. Various criteria must be taken into account to ensure error-free binding to the starting DNA. It is therefore not uncommon for several variants to be created and tested in practice.

In the next step, the polymerase can then bind to these primers and start replicating the DNA.

The process of actual amplification is called extension, elongation or amplification. Here, DNA polymerase migrates the nucleotides of the DNA single strand piece by piece. Meanwhile, it creates a complementary counterpart to the original strand with the help of free nucleotides (deoxy nucleotides = dATP, dGTP, dCTP, dTTP). This is therefore an identical copy of the other single strand.

Each of these processes has a particular temperature. This is the reason why a thermostable DNA polymerase is used like for example the Pfu- or Tag-Polymerase. Furthermore, a constant change depending on the reaction step and cycle is not possible manually, therefore a PCR is performed in a so-called thermal cycler. As the name already describes, this is a PCR machine that runs through different temperatures in defined cycles.

 

A selection of our DNA polymerases

 

Product Supplier Art. No. Amount
TAQ DNA POLYMERASE

GenScript

E00007-1000
E00007-50000

1000.0U
50000.0U

 
RECOMBINANT TAQ DNA POLYMERASE

Prospec

enz-308-1,000U
enz-308-3,000U
enz-308-10,000U

1,000U
3,000U
10,000U

 
EXPRIME TAQ DNA POLYMERASE (WITH DNTP MIXTURE AND 10X RXN BUFFER (WITH MG))

BioBiz

G-1000

1,000U

 
HS PRIME TAQ DNA POLYMERASE (WITH 10MM DNTP MIX., 25MM MGCL2 & 10X RXN BUFFER(MG FREE))

BioBiz

G-7000XX

250 Units

 
TAQ DNA POLYMERASE (RECOMBINANT)

Abbexa

abx073521-1000
abx073521-3000
abx073521-10000

1,000U
3,000U
10,000U

 
TAQ DNA POLYMERASE, 5U/­UL

Bio Basic

BB-B0089-200
BB-B0089-500
BB-B0089-5x200
BB-B0089-1
BB-B0089-5x1000

200U
500U
5x200U
1000U
5x1000U

 
FAST-­TAQ DNA POLYMERASE WITH 10MM DNTP MIX

Bio Basic

BB-9K-001-0003
BB-9K-001-0004

500U
2500U

 
HIGH-­TAQ DNA POLYMERASE WITH 10MM DNTP MIX

Bio Basic

BB-9K-001-0005
BB-9K-001-0005

250U
4x250U

 
AT TAQ DNA POLYMERASE (HOTSTART -­ 5U/­UL)

Pop-Bio

POV-PL3201
POV-PL3202

200U
500U

 
HOTSTART TAQ DNA POLYMERASE (5U/­UL)

Biomatik

BM-A4144-250U

250U

 

 

To make the procedure of a PCR as simple as possible a PCR Mastermix is often used. Such a PCR mastermix contains the DNA polymerase, a suitable reaction buffer, as well as the required nucleotides, which serve as building blocks for the new "copies".

 

A selection of our PCR master mixes

 

Product Supplier Art. No. Amount
EZWAY MULTIPLEX PCR MASTERMIX (2X)

Komabiotech

K0567810

1 ml

 
EZWAY MULTIPLEX PCR QMASTERMIX (2X)

Komabiotech

K0567820

1 ml

 
2X HOTSTART TAG PCR MASTERMIX WITH LOADING DYE

Tools Biotech

BTL-KTT-BB02

1 ml

 
EXPRESS HI FI DNA POL, 2X MASTERMIX

Biomiga

BMG-EHF1102-01

BMG-EHF1102-02

2,5 ml (100 Reactions)
12,5 ml (500 Reactions)

 
VIPRIMEPLUS ONESTEP QRT-­PCR MASTERMIX

Pop-Bio

POV-QLMM03

1ea

 
GENEKAM PCR MASTERMIX SOLUTION (2X) 1000 REACTIONS

Genekam-Biotechnology

GEK-SB0012

10 ml

 

 

Use of a PCR test to detect diseases

 

The PCR method can be advantageous not only for the amplification of DNA for an upcoming experiment.

Hereditary diseases are caused by changes in the genome. The specific gene can be amplified by means of PCR. Subsequently, the material is sequenced to detect possible mutations.

A PCR test can also help in the diagnosis of viral diseases such as an infection with the HI virus. In this test the viral DNA is amplified and can then be examined. In the case of RNA viruses, the RNA is transcribed into cDNA by a reverse transcriptase. This allows amplification by PCR as described above (RT-PCR).

 

HIV-PCR Test

 

A PCR test can also be used for the detection of HIV. It offers a central difference to other tests: While other tests detect antibodies, an HIV PCR test detects the HIV virus.

However, it should be emphasized that this HIV-PCR test is more likely to be used in the context of therapy or in connection with an antibody screening test than as a control test. Here, the previously obtained result should only be confirmed again.

 

Reverse transcriptase polymerase chain reaction

 

Similar to PCR, the reverse transcriptase polymerase chain reaction (RT-PCR) is divided into three steps: Purification of the RNA, reverse transcription of the RNA into DNA and amplification of the cDNA thus generated.

While we have previously learned about polymerase, this variant of PCR is followed by the introduction of another enzyme: reverse transcriptase. This enzyme originally comes from retroviruses. However, this enzyme is further modified for industrial use. For example, its RNase H activity has usually been removed.

The reverse transcriptase is also a DNA polymerase. However, the difference to the "normal" polymerase is that the reverse transcriptase requires RNA as starting material. It is therefore also known as RNA-dependent DNA polymerase. Using a DNA primer, the reverse transcriptase is able to produce complementary DNA (cDNA).

The difference between DNA and cDNA is that the latter has no introns. This is due to the fact that cDNA is produced by means of an RNA strand that has already gone through the splicing process.

 

A selection of our products for RT-PCR

 

Products Supplier Art. No. Amount
One-­Step RT-­PCR Kit

Biomiga

BMG-RT0201-00
BMG-RT0201-01
BMG-RT0201-02

5 Reactions
25 Reactions
100 Reactions

 
One Step RT PCR Kit (cDNA Synthesis + PCR In One Reaction)

Boster

BOS-MB1003

100 Reactions

 
GENEKAM RT MASTER MIX

Genekam Biotechnology

GEK-K272

100 Reactions

 

 

Real Time Quantitative PCR

 

Real Time Quantitative PCR (qPCR for short) is a common PCR in which the synthesized DNA is additionally quantified. The term "real time" refers to the quantification process: the increase in material is measured in real time using fluorescence.

One possibility is, for example, the use of DNA dyes. Commonly used are ethidium bromide or SYBR Green I. After successful synthesis of the complementary strand, the starting material is again available as a double-helix strand. The corresponding dyes can then intercalate into the DNA and thus stain it. The increase in synthesized material is proportional to the fluorescence intensity of the reaction batch.

 

A selection of our products for Real Time Quantitative PCR

 

Products Supplier Art. No. Amount
SYBR GREEN I NUCLEIC ACID GEL STAIN

ChemScene

CS-0084765-100ug
CS-0084765-500ug

100 µg
500 µg

 
Prime Q-­Master Mix (2X conc. with SYBR Green I)

BioBiz

Q-9200

100 ml

 
SUPRIMESCRIPT QRT-­PCR KIT (FOR TAQMAN PROBE, 2X CONC., WITH ROX DYE)

BioBiz

Q-5100

100 Reactions

 
SYBR GREEN I NUCLEIC ACID GEL STAIN

MedChem Express

HY-K1004-100uL
HY-K1004-500uL

100 µl
500 µl

 
RT MASTER MIX FOR QPCR

MedChem Express

HY-K0510-1mL
HY-K0510-5mL

1 ml (100 Reactions)
5 ml (5x 100 Reactions)

 
KOD SYBR QPCR MIX

Cosmobio

TYB-QKD-201T
TYB-QKD-201

1 ml
3*1.67 ml

 
FAST EVAGREEN MASTER MIX FOR QPCR(200 RXN)

Biotium

B-31003-T
B-31003
B-31003-1
B-31003-2

1 ml
2*1 ml
5*1 ml
50*1 ml

 
CYBRFAST 1-­STEP RT-­QPCR HI-­ROX KIT

Tonbo Biosciences

31-5202-0100R

100 Reactions

 
VALIDATED FLUORESCENT PROBES FOR REAL-­TIME QUANTITATIVE PCR (TAQMAN PROBE: FAM-­BHQ1)

GenePharma

GP-F01001

200 Reactions

 

 

Real time quantitative PCR for the detection of SARS-CoV-2

 

In view of the rapid spread of SARS-CoV-2, the demand for direct and indirect detection methods for COVID-19 has increased rapidly worldwide. Indirect detection methods include serological tests based on either SARS-CoV-2 antibody detection or SARS-CoV-2 antigen detection. In most cases, however, such tests must be combined with direct virus detection. RT-qPCR is the most widely used test.

 

A selection of our products for the detection of SARS-CoV-2 by RT-qPCR

 

Products Supplier Art. No. Tests/RXNs
Novel Coronavirus 2020 (Wuhan Strain specific) Realtime PCR

Genekam Biotechnology

GEK-FR475

100 Tests

SARS-­CoV-­2 qPCR detection 1-­plex assay-­ORF1ab
SARS-­CoV-­2 qPCR detection 1-­plex assay-­N
SARS-­CoV-­2 qPCR detection 1-­plex assay-­RdRP
SARS-­CoV-­2 qPCR detection 1-­plex assay-­E

GenScript

SC1618-ORF1ab
SC1618-N
SC1618-RdRP
SC1618-E

100 rxns
100 rxns
100 rxns
100 rxns

ABScript II One Step RT-­qPCR Probe Kit

Abclonal

RK20407-20rxn
RK20407-100rxn

20 rxns
100rxns

Coronavirus (SARS-­CoV-­2) Real Time RT-­PCR Nucleic Acid Detection Kit

Raybiotech

PCR-COV

20 Tests

COVID-­19 RT-­qPCR Rapid Detection Kit

ABM

ABM-G628

100 rxns

 

Possible PCR error sources

 

There are many things that can (but need not) go wrong with a PCR.

For instance, the correct concentration of the individual reagents plays an important role in most cases. Special attention should be paid to the dNTPS, the MgCl solution, the polymerase and the template.

 

TIP: Especially as a newcomer to the laboratory it helps to exchange information with colleagues and ask for their standard PCR protocol. These are usually long established. If the opportunity arises, you might even be able to use their preset thermal cycler program.

 

An incorrect thermal cycler program can also be a possible source of PCR errors.

However, if the DNA polymerase does not start working at all, no material can be amplified in a PCR. This might be due to a faulty primer design, for example.

 

TIP: It is recommended to synthesize several primer pairs at the beginning of the experiment. So you always have a back-up if one pair does not work and if all of them bind you have an additional control.

 

Frequently asked questions:

 

What is a PCR test?

A PCR test is a detection test in which the presence of a specific DNA (or RNA) is shown by its amplification.

 

What is PCR diagnostics?

In diagnostics, for example, a PCR test can be used to detect an underlying genetic defect.

 

How long does a PCR test take?

The duration of a PCRT test depends on the number of cycles used.

 

Who invented PCR?

Kary Mullis is the inventor of PCR.

 

What is a primer in biology?

A primer is an oligonucleotide. This single-stranded DNA fragment serves as a starting point for DNA polymerase and is specifically used within a PCR.

 

Which enzyme removes the primers?

The 5'-3' exonuclease activity of Polymerase I or RNase H removes primers in prokaryotes.

 

Why are there Okazaki fragments?

Due to the 5'-->3' synthesis activity of DNA polymerase, discontinuous small DNA fragments are generated on the subsequent strand (complementary to the leader strand). These sections are called Okazaki fragments.