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0Isothermal amplification technology can achieve the purpose of amplifying specific nucleic acid sequences under constant temperature conditions. Due to the characteristics of no special equipment, short amplification time, and high sensitivity, it has shown good application prospects in on-site detection and immediate diagnosis. Suitable for the development of molecular.
1. Diagnostic tools.
2. High-quality isothermal amplification core enzyme raw materials.
3. Product selection guide.
1. Diagnostic tools.
According to different amplification principles, Isothermal nucleic acid amplification techniques can be divided into many categories according to different amplification principles, including Loop-Mediated Isothermal Amplification (LAMP), Cross-Primer Amplification (CPA), Strand Displacement Amplification (SDA), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequence Based amplification (NASBA), Rolling Loop Amplification (RCA) and Helicase-Dependent Amplification (HAD), etc. At present, LAMP is the most common and widely used isothermal amplification technology that developed by Japanese scholar Notomi in 2000. The technical feature of LAMP is to design four primers for six regions of the target genes, and Bst DNA polymerase is used to perform amplification reaction at a constant temperature of 60~65℃, and achieve 109-10 fold amplification within 15-60 min.
2. High-quality isothermal amplification core enzyme raw materials
For LAMP/RT-LAMP technology, our company can provide high-quality isothermal amplification core enzyme raw materials, including strand displacement Bst DNA polymerase, MMLV reverse transcriptase, UDG enzyme, and RNase inhibitors. It has been praised by many customers, perfectly replaced the imported brands in use, and solved the problems of delivery time and cost for customers.
2.1 Yeasen- Bst DNA Polymerase
Yeasen Hieff™ Bst Plus DNA Polymerase (Cat#14402ES, 14403ES) is obtained by expressing and purifying the DNA polymerase gene of Bacillus stearothermophilus sp lacking the 5′→3′ exonuclease domain in E. coli. The enzyme strand displacement ability is strong, and has the advantages of high sensitivity, high amplification efficiency, and high dUTP tolerance, and can be widely used in the real-time detection of pathogens based on isothermal amplification technology.
2.1.1 The sensitivity is as low as 50 copies/T, and the reaction time is less than 15 minutes (fluorescence quantitative RT-LAMP method)
Figure 1. RT-LAMP reaction with Hieff™ Bst Plus DNA Polymerase to amplify SARS-CoV-2. In the 25 μL reaction system, the detection rate of 50 copies/T reached 100% , and the reaction time was less than 15 min.
2.1.2 High dUTP tolerance
Yeasen Hieff™ Bst Plus DNA Polymerase for RT-LAMP reaction to amplify SARS-CoV-2 (20 copies/T). The dUTP/UDG enzymatic anti-fouling system was introduced in the recommended reaction scheme, and dUTP was used to replace dTTP. Comparing the amplification results of 35 mM dUTP replacement ( red amplification curve) and T: U = 1:1 ( blue amplification curve), it was found that the addition of dUTP in the reaction system had no effect on the sensitivity and amplification efficiency. The enzyme has high dUTP tolerance.
Figure 2. The results of the high dUTP tolerance test of Yeasen Hieff™ Bst Plus DNA Polymerase.
2.2 Yeasen -MMLV Reverse Transcriptase
Yeasen Hifair™ Ⅲ Reverse Transcriptase (Cat #11111ES, 14601ES) has fast cDNA synthesis speed, high thermal stability, and can tolerate reaction temperatures up to 65°C. It is suitable for reverse transcription of RNA templates with complex secondary structures. At the same time, the enzyme enhances the affinity with the template, which is very suitable for the reverse transcription of a small number of templates and low-copy genes. In addition, this product can amplify up to 19.8kb cDNA.
2.2.1 The recommended reaction temperature is 55℃, for high GC content templates or complex templates, reverse transcription temperature can be increased to 60°C.
Figure 3. Yeasen Hifair ™ III Reverse Transcriptase (Cat#11111ES) at 42-65°C using 300 ng of 297T cell total RNA as template. Take 1 μL of cDNA as the template, and use 2×Hieff Canace™ Gold PCR Master Mix (Cat#10149ES) to amplify different types of templates.
2.3 Yeasen- UDG enzyme
Yeasen Uracil DNA Glycosylase (UDG/UNG), 1 U/μL (Cat #14455ES) can catalyze the hydrolysis of the N-glycosidic link between the uracil base and the sugar-phosphate backbone in ssDNA and dsDNA. It can easily control aerosol pollution and is suitable for common molecular biology systems such as PCR, qPCR, and RT-qPCR.
2.3.1 Strong digestive ability: UDG enzyme (0.025 U) fully digests 360 ng of 200 bp dU -DNA.
Figure 4. Electrophoresis results of 0.05 U, 0.025 U, 0.0125 U, 0.00625 U, 0.003125 U anti- contamination UDG enzyme with 360 ng of 200 bp dU -DNA incubated at 25°C for 30 min.
2.3.2 Strong compatibility: perfectly compatible with downstream qPCR and RT-qPCR reaction systems, and has no impact on the detection system under high input.
Figure 5. Different concentrations of UDG enzymes are compatible with qPCR and RT-qPCR reaction systems and have no inhibition on the amplification of DNA and RNA templates.
2.4 Yeasen-RNase Inhibitor
Yeasen Murine RNase Inhibitor (Cat #10603ES, 10610ES) is a recombinant murine RNase inhibitor expressed in soluble form in E. coli that can inhibit a wide range of RNase (RNase A, B, C) and can be used for RT-PCR/RT-qPCR, solving the problem of RNA degradation in vitro transcription, and it can compatible with various reverse transcriptases and DNA polymerases.
2.4.1 Highly efficient RNase inhibitory ability
Figure 6. 1μL Murine RNase inhibitor ( 40 U/μL, Cat#10603ES ) can effectively inhibit 5ng RNase, 1μg HEK cell total RNA electrophoresis results.
2.4.2 Murine RNase Inhibitor outperforms international similar products in qPCR experiments
Table 1. Different concentration gradients of MRI were incubated with 100 ng of RNase A to block the activity of RNase A, followed by digestion with 1 μg of RNA. One - step RT-qPCR was used to detect the degradation of RNA to judge the blocking effect of MRI on RNase A. PC representative system only RNA without RNase A and MRI
|
MRI |
80U |
60U |
40U |
30U |
20U |
10U |
0U |
PC |
|
Yeasen Ct |
12.51 |
12.5 |
12.96 |
13.12 |
17.22 |
29.64 |
39.12 |
11.23 |
|
R*Ct |
|
|
14.09 |
13.84 |
14.36 |
27.58 |
- |
- |
|
∆Ct |
|
|
1.13 |
0.68 |
﹣2.86 |
﹣2.06 |
- |
- |
3. Product Selection Guide
The products provided by Yeasen are as follows.
Table 2. Product selection guide
|
Product Positioning |
product name |
Cat# |
|
Highly sensitive Bst enzyme |
14402ES |
|
|
Hieff™ Bst Plus DNA Polymerase (2,000 U/μL) (Inquire) |
14403ES |
|
|
Reverse Transcriptase for RT-Lamp |
Hifair™ Ⅲ Reverse Transcriptase (200 U/μL) (Inquire) |
11111ES |
|
Hifair™ Ⅲ Reverse Transcriptase (600 U/μL, Glycerol-free) (Inquire) |
11297ES |
|
|
RNase inhibitor |
10603ES |
|
|
Murine RNase inhibitor (200 U/μL) (Inquire) |
10610ES |
|
|
Murine RNase Inhibitor (200 U/µL, Glycerol-free) (Inquire) |
10703ES |
|
|
UDG/UNG |
Uracil DNA Glycosylase (UDG/UNG), 1 U/μL (Inquire) |
14455ES |
|
RT-LAMP Kit (Dye Assay) |
RT-LAMP Dye Assay Kit (UDG plus) (Inquire) |
13762ES |
|
RT-LAMP Kit (pH Sensitive Dyestuff) |
RT-LAMP pH Sensitive Dyestuff Kit (Inquire) |
13906ES |
|
dUTP with high purity |
10128ES |
|
|
dNTP Mix with high purity |
10125ES |
