Blood Direct PCR Kit

Rapid amplification of the target gene directly using blood as a template without extraction.

This kit adopts a genetically engineered anti-inhibitor DNA polymerase to efficiently amplify single-copy genes in the human genome. The well optimized buffer system in this kit helps the polymerase strongly resist the inhibition of PCR inhibitors, thus can directly amplify DNA using blood and cultured cells as templates. This product is easy to operate and does not require complicated steps such as DNA purification or sample pretreatment.
This kit is provided as 2×MasterMix, and the reaction can be performed by simply adding the blood template and corresponding detection primers. It can be applied on the cultured cells of mammals such as humans, mice, pigs, cattle and other species, as well as fresh or 4℃ cryopreserved whole blood, anticoagulant (EDTA, citrate, heparin), liquefied blood clots and dry blood spots stored on Whatman 903 and FTA Elute commercial cards.

Cat. No	Packing Size
4992529	20 µl×100 rxn
4992530	20 µl×500 rxn

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Product Detail

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Features

■ Simple and fast: PCR amplification can be directly performed using blood as template, without the need for the tedious steps of sample preparation and DNA extraction.
■ High purity: The skip of sample pre-treatment and DNA extraction steps can help to avoid the cross-contamination of samples.
■ High throughput: The PCR identification for large-scale samples can be performed by combining the kit with 96/384-well PCR plates.
■ Strong universality: This kit can efficiently amplify high GC fragments or fragments with complex secondary structure, and the amplification length can be up to 5 kb.
■ Strong stress resistance: This kit can be applied for various species and blood samples preserved in different ways.

Applications

The PCR products of this kit contains “A” at the 3′-end, which can be directly used for TA vector cloning. This kit can be used for the amplification of genomic DNA fragments, high-throughput genetic analysis and genotyping (such as gene detection) analysis.

All the products can be customized for ODM/OEM. For details, please click Customized Service(ODM/OEM)

Experimental Example

	Using human EDTA anticoagulation as a template, 4 genes with different GC contents were amplified by Blood Direct PCR Kit . The PCR reaction system was 20 μl, and 1 μl blood was used as template. M: TIANGEN Marker II; 1: Fragment size 1090 bp, GC content 68.1%; 2: Fragment size 1915 bp, GC content 70.4%; 3: Fragment size 448 bp, GC content 74.8%; 4: Fragment size 1527 bp, GC content 61.5%. Experimental results: Blood Direct PCR Kit can effectively amplify DNA fragments with the GC content at the range of 61.5%-74.8%, suggesting it’s capable to amplify high-GC fragments.
	Using human EDTA anticoagulation as template, 5 genes with different lengths (ActB, Prp, DN1.0, Hn2.0 and Hn4.0) were amplified by Blood Direct PCR Kit. The PCR reaction system was 20 μl, and 1 μl blood was used as template. M: TIANGEN Marker II; 1-3: 3 different blood samples; NTC: control without primers. Experimental results: Blood Direct PCR Kit can amplify fragments with the length as long as 4 kb, suggesting it’s capable to amplify long fragments.
	Using human EDTA anticoagulation as template, Blood Direct PCR Kit was used for PCR detection of different blood samples. The PCR reaction system was 20 μl, and 1 μl blood was used as template. M: TIANGEN Marker II; 1-9: the loading amount of blood is 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl and 5 μl, respectively; NTC: control with no template Experimental results: Blood Direct PCR Kit has strong resistance to blood and can amplify blood samples with the loading range of 0.1-5 μl.
	Blood samples from human, rat, chicken and other species with different treatments were used as templates. Blood Direct PCR Kit was used to amplify PRNP (human, 750 bp), Actin (rat, 200 bp), and β-Actin (Chicken, 1.0 kb). The PCR reaction system was 20 μl, and 1 μl blood was used as template. M: TIANGEN Marker II. Experimental results: Blood Direct PCR Kit can be applied on a wide range of samples, and the direct PCR detection can be performed on blood samples from various species with different treatments.

FAQ

Q: No amplification bands

A-1 Template

■ The template contains protein impurities or Taq inhibitors, etc. ——Purify DNA template, remove protein impurities or extract template DNA with purification kits.

■ The denaturation of template is not complete ——Appropriately increase denaturation temperature and prolong denaturation time.

■ Template degradation ——Re-prepare the template.

A-2 Primer

■ Poor quality of primers ——Re-synthesize the primer.

■ Primer degradation ——Aliquot the high concentration primers into small volume for preservation. Avoid multiple freezing and thawing or long-term 4°C cryopreserved.

■ Inproper design of primers (e.g. primer length not sufficient, dimer formed between primers, etc.) -Redesign primers (avoid formation of primer dimer and secondary structure)

A-3 Mg²⁺concentration

■ Mg²⁺ concentration is too low ——Properly increase Mg²⁺concentration: Optimize the Mg²⁺ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg²⁺ concentration for each template and primer.

A-4 Annealing temperature

■ The high annealing temperature affects the binding of primer and template. ——Reduce the annealing temperature and optimize the condition with a gradient of 2°C.

A-5 Extension time

■ Short extension time——Increase extension time.

Q: False positive

Phenomena: Negative samples also show the target sequence bands.

A-1 Contamination of PCR

■ Cross contamination of target sequence or amplification products ——Carefully not to pipet the sample containing target sequence in the negative sample or spill them out of the centrifuge tube. The reagents or equipment should be autoclaved to eliminate existing nucleic acids, and the existence of contamination should be determined through negative control experiments.

■ Reagent contamination ——Aliquot the reagents and store at low temperature.

A-2 Primer

■ Mg²⁺ concentration is too low ——Properly increase Mg²⁺ concentration: Optimize the Mg²⁺ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg²⁺ concentration for each template and primer.

■ Improper primer design, and the target sequence has homology with the non-target sequence. ——Re-design primers.

Q: Non-specific amplification

Phenomena: The PCR amplification bands are inconsistent with the expected size, either large or small, or sometimes both specific amplification bands and non-specific amplification bands occur.

A-1 Primer

■ Poor primer specificity

——Re-design primer.

■ The primer concentration is too high ——Properly increase the denaturation temperature and prolong denaturation time.

A-2 Mg²⁺ concentration

■ The Mg²⁺ concentration is too high ——Properly reduce the Mg2+ concentration: Optimize the Mg²⁺ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg²⁺ concentration for each template and primer.

A-3 Thermostable polymerase

■ Excessive enzyme amount ——Reduce enzyme amount appropriately at intervals of 0.5 U.

A-4 Annealing temperature

■ The annealing temperature is too low ——Appropriately increase the annealing temperature or adopt the two-stage annealing method

A-5 PCR cycles

■ Too many PCR cycles ——Reduce the number of PCR cycles.

Q: Patchy or smear bands

A-1 Primer ——Poor specificity ——Re-design the primer, change the position and length of the primer to enhance its specificity; or perform nested PCR.

A-2 Template DNA

——The template is not pure ——Purify the template or extract DNA with purification kits.

A-3 Mg²⁺ concentration

——Mg²⁺ concentration is too high ——Properly reduce Mg²⁺ concentration: Optimize the Mg²⁺ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg²⁺ concentration for each template and primer.

A-4 dNTP

——The concentration of dNTPs are too high ——Reduce the concentration of dNTP appropriately

A-5 Annealing temperature

——Too low annealing temperature ——Appropriately increase the annealing temperature

A-6 Cycles

——Too many cycles ——Optimize the cycle number

Q: How much template DNA should be added in a 50 μl PCR reaction system?

Q: How to amplify long fragments?

The first step is to choose the appropriate polymerase. Regular Taq polymerase cannot proofread due to lack of 3’-5’ exonuclease activity, and mismatch will greatly reduce the extension efficiency of fragments. Therefore, regular Taq polymerase cannot effectively amplify target fragments larger than 5 kb. Taq polymerase with special modification or other high fidelity polymerase should be selected to improve extension efficiency and meet the needs of long fragment amplification. In addition, the amplification of long fragments also requires corresponding adjustment of primer design, denaturation time, extension time, buffer pH, etc. Usually, primers with 18-24 bp can lead to better yield. In order to prevent template damage, the denaturation time at 94°C should be reduced to 30 sec or less per cycle, and the time to rise temperature to 94°C before amplification should be less than 1 min. Moreover, setting the extension temperature at about 68°C and designing the extension time according to the rate of 1 kb/min can ensure effective amplification of long fragments.

Q: How to improve the amplification fidelity of PCR?

The error rate of PCR amplification can be reduced by using various DNA polymerases with high fidelity. Among all the Taq DNA polymerases found so far, Pfu enzyme has the lowest error rate and the highest fidelity (see attached table). In addition to enzyme selection, researchers can further reduce PCR mutation rate by optimizing reaction conditions, including optimizing buffer composition, concentration of thermostable polymerase and optimizing PCR cycle number.

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