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Cat. No.: HMMDKI-8 (for 8T)

Cat. No.: HMMDKI-96 (for 96T)

Description

Human mtDNA Mutation Detection Kit (For Illumina) is a high-throughput sequencing library construction kit used for genotyping detection of the entire gene length of human mitochondrial DNA (hmtDNA). This kit employs multiple PCR reactions to amplify and enrich the full sequence of human mitochondrial DNA, and utilizes Bidirect Amp Dual-Index Primers (For Illumina) to add dual-end tags to each DNA sample, thereby stably and efficiently obtaining mtDNA amplicon sequencing libraries suitable for the Illumina sequencing platform, ultimately obtaining mutation information for the entire length of human mitochondrial DNA.

Mitochondria are the powerhouses of the cell, being the primary sites for cellular redox reactions. Structural and functional abnormalities in mitochondria can lead to the onset of various diseases. Each mitochondrion contains approximately 2-10 copies of mtDNA, usually located near the inner membrane or in the matrix. mtDNA is circular double-stranded and typically 16,569 base pairs long, divided into control and coding regions. The control region is a high mutation rate region of mitochondrial DNA, while the coding region encodes 37 genes, including 13 polypeptides, 22 tRNAs, and 2 rRNAs. The main function of the proteins encoded by mtDNA is to regulate oxidative phosphorylation and protein assembly, and mtDNA mutations are the primary cause of structural and functional abnormalities.

Diseases caused by mitochondrial mutations are known as mitochondrial genetic disorders, with an incidence rate of about 1 in 5,000. Currently, over 100 human diseases have been found to be associated with mitochondrial gene mutations, such as Leber's hereditary optic neuropathy, mtDNA mutations associated with aminoglycoside-induced deafness (also known as "one-shot deafness"), MELAS syndrome, MERRF syndrome, Kearns-Sayre syndrome, etc. Mitochondrial genetic diseases exhibit maternal inheritance and threshold effects.

Mitochondrial mutation detection can assist in the early diagnosis of mitochondrial genetic diseases, which is crucial for clarifying symptoms and ensuring good prognosis for patients. Additionally, mitochondrial mutation detection can effectively prevent the occurrence of "one-shot deafness." Therefore, the detection and research of human mitochondrial DNA mutations are very important.

This kit contains primers and reagents for multiple PCR amplifications for mitochondrial mutation detection, Primer Cleanup Mix for removing excess primers, and relevant reagents for labeling PCR reactions for each sample. Primers targeting regions with universal labels bind to mitochondrial genome DNA, and through multiple PCR reactions, the entire mitochondrial genome DNA is amplified in multiple segments. After mixing the amplification products of the same sample, they are digested with Primer Cleanup Mix, diluted 1:100, and then subjected to a second round of labeling amplification reactions for positive and negative strands using Bidirect Amp Dual-Index Primers (For Illumina) to add sequencing tags to the samples. Finally, sequencing library construction is completed after magnetic bead purification.

This kit does not include PCR primers for adding library tags. It is recommended to purchase Bidirect Amp Dual-Index Primers (For Illumina) for this purpose.

The main effective component of Primer Cleanup Mix is single-strand nuclease, which is used to digest and remove excess primers after PCR reactions, reducing the negative impact of excess primers on the second PCR.

This kit is suitable for genomic DNA derived from blood, saliva, or other tissues, but only applicable to point mutations and small deletions/insertions mutations within 20 base pairs (micro-mutations).

Human mtDNA Mutation Detection Kit (For Illumina) is available in two different sizes of packaging, suitable for users with 384-well PCR instruments, for the construction of mtDNA full-length amplicon sequencing libraries for 8 and 96 samples respectively. For users using PCR tubes or 96-well PCR systems, it is recommended to double the volume of the first multiplex PCR reaction to 10 μl, which can effectively reduce the failure rate of amplification caused by volume loss.

Components

• hmtDNA ProbeMix01
• hmtDNA ProbeMix02
• hmtDNA ProbeMix03
• hmtDNA ProbeMix04
• Multiplex PCR Mix
• Primer Cleanup Mix
• Index PCR Master Mix

Storage

Store at -20°C, valid for at least one year.

Notes

• Reagents and materials to be provided by the user: Bidirect Amp Dual-Index Primers (For Illumina), DNA Fragment Selection Magnetic Beads, ethanol, Ultrapure Water (DNase/RNase-Free, Sterile), Magnetic Separators, 96/384-well PCR instrument, 96-well or 384-well PCR plates, heat-resistant PCR sealing film.
• Multiplex PCR Mix, Primer Cleanup Mix, and Index PCR Master Mix contain enzymes that are prone to inactivation. They should be stored in a cooler or on ice during use and immediately placed at -20°C after use.
• This product is intended for scientific research by professionals only and is not intended for clinical diagnosis or treatment, food, or drugs, and should not be stored in ordinary residential areas.
• For your safety and health, please wear lab coats and disposable gloves during operation.

Only for research and not intended for treatment of humans or animals

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