Features

Kit for fast genotyping from human and animal blood samples and blood dried on blood cards

• Direct blood handling with the Blood Transfer Tool and DNA preparation within 1 minute
• Inhibitor Removal Pearls for superior PCR results for challenging blood samples
• No step for lysis, disruption, or dilution of blood samples required

 

Technology	Direct HotStart PCR for blood sample material
Format	10 μL PCR (optional up to 50 μL)
Sample material	Whole blood from e.g., human, mouse, rat, cat, chicken, rabbit, guinea pig, sheep, or cow treated with EDTA, citrate, or heparin. Punches from blood storage cards (e.g., NucleoCard® or FTA).
Procedure	Pretreatment of blood with 1 Inhibitor Removal Pearl for 50–500 μL blood (optional) and transfer of blood aliquot with the Blood Transfer Tool into PCR mix
PCR	Use the NucleoType HotStart PCR Master Mix for fast PCR genotyping.
Amplicon size	Recommended for up to 1 kb
Preparation time	< 1 min; PCR cycling: 30–90 min (cycler and target size dependent)
Stability	12 months at 4 °C or -20 °C

Applications*

• Direct HotStart PCR analysis from human and animal blood samples

* Kits to be used for research purposes only

Principle / Procedure

Many blood genotyping methods are based on DNA purification out of whole blood, followed by PCR amplification of genes of interest. However, DNA purification from whole blood is a time consuming and elaborate process. No special step for lysis or disruption of blood samples is required for the NucleoType kit: A standardized blood aliquot from liquid blood sample material is directly applied to the PCR with the Blood Transfer Tool (BTT) (provided in the kit). This procedure enables easy and fast genotyping for many different kinds of blood samples (e.g., human, cat, lamb, cattle).

Some blood types which are even more challenging e.g., in avian blood such as chicken, the mature red cells obtain a nucleus. Therefore, the blood contains hundred times more DNA and PCR inhibition is a well known issue. Such blood types can also easily be processed by the addition of 1 Inhibitor Removal Pearl (provided in the kit) to max. 500 µL blood sample before sampling a blood aliquot via BTT for PCR. Furthermore, the enzyme mix enables cycling times of less than one hour. Analysis of the amplicons can be performed by gel electrophoresis or Bioanalyzer®. The reactions contain a loading dye and are ready to apply directly on the agarose gel.

In order to obtain reliable blood genotyping data, it is important to transfer just a trace amount of blood to obtain DNA in a form suitable to serve as template for subsequent PCR amplification. Common Blood PCR kits on the market are either diluting the blood sample, or need a centrifugation/neutralization step before or after the PCR reaction.

The NucleoType Blood PCR kit contains Blood Transfer Tools (BTT) suitable to transfer the blood from the sample into the PCR mix. Touching the blood surface with the pinpoint end of the Blood Transfer Tool (approx. 1–2 mm deep into the blood) is sufficient to obtain superior PCR results. In addition to the Blood Transfer Tools, the kit contains Inhibitor Removal Pearl (IRP) for challenging blood sample materials. The procedure is fairly simple by adding 1 IRP to 50–500 μL blood aliquots, brief mixing (e.g. by vortexing) and incubation for ~15 seconds. Afterwards the blood can be transferred to the PCR Mix using the Blood Transfer Tool.