SAFM™ Technology

SAFM™ Technology (patent pending) refers to Selective Amplification of Family Members including all known and novel members of gene families. This technology is particularly designed to fundamentally overcome the current inherent problems and limitations of degenerate PCR by utilising Seegene's proprietary ACP™ (Annealing Control Primer) system.

The ACP system, a process to maximise PCR specificity by using primers that anneal specifically to the template, is incorporated in the SAFM™ by means of a degenerate ACP system and degenerate nested ACP-PCR. This gives the following advantages:

• The degenerate ACP system provides a high tolerance in "primer search parameters" such as degeneracy, the length of degenerate sequence, the melting temperature of the degenerate sequence and GC content. It also allows a high annealing temperature, at least 10 to 30°C higher.

• Degenerate nested ACP-PCR dramatically improves specificity and sensitivity. The nested ACP system is prevented from nonspecific binding and the results are sensitive enough to detect rare transcripts which would be undetectable even by the conventional RT-PCR.

• Overlapping sequences of the degenerate nested ACP-PCR cover a large range of amino acid sequences of the conserved domain. This results in isolating only members of a gene family, such as a homeobox family.

Key Features of SAFM™

1. The degenerate ACP system provides a high tolerance in "primer search parameters" such as degeneracy, the length of degenerate sequence, the melting temperature of the degenerate sequence and GC content.

2. The degenerate ACP system allows for a high annealing temperature, for example, at least 10 to 30°C higher than the melting temperature of the degenerate sequence.

3. Degenerate nested ACP-PCR improves specificity and sensitivity.

• Specificity: SAFM™ Technology overcomes the problem of nonspecific amplification resulting from the nonspecific binding of the nested primer, which is the major hurdle of current conventional nested PCR.

• Sensitivity: SAFM™ Technology is sensitive enough to detect rare transcripts which are not detectable even by the conventional RT-PCR.

• Gene Family Mass Differential Analysis: So far, there is no method or comparable commercialised kit for the differential analysis of all members of a homeobox family.

4. Overlapping nested ACP-PCR generates a large range of amino acid sequences of the conserved domain, which is sufficient enough to isolate only members of a gene family such as a homeobox family.

5. Degenerate nested ACP systems reduces degeneracy and the number of nested primers, which saves a significant amount of time and cost.

Why SAFM™ Technology is World's First?

• Specificity of Gene Family Discovery:
  Exclusively amplifying all known and novel members of gene families such as a homeobox family within 1 day.

• Sensitivity of Gene Family Discovery:
  Sensitive enough to detect rare transcripts which are not detectable even by the conventional RT-PCR.

• Mass Differential Expression Analysis of Gene Families:
  Ideal for the differential analysis of all members of a homeobox family expressed in your target cells.

• Gene Family Mass Screening:
  So far, there is no method or comparable commercialised product due to the limitations of current available approach methods.

• Detection of Rare Transcripts:
  The SAFM™ detection system is at least 1 billion times more sensitive than any available RT-PCR or Real-time PCR method. So far, there is no method or comparable commercialised product due to the limitations of current available approach methods.

• Gene Family Mass Differential Analysis:
  So far, there is no method or comparable commercialised kit for the differential analysis of all members of a homeobox family.

Current Problems of Degenerate (RT-)PCR

1. Degenerate (RT-)PCR

A common way to isolate members of a gene family from either cDNA or genomic DNA is to use PCR with degenerate primers (mixtures of primers), designed on the basis of a conserved region (domain) sequence data from alignment of gene families.

2. Inherent problems in degenerate (RT-)PCR

• Degenerate primer design is very difficult due to codon degeneracy and the additional degeneracy needed to represent multiple codons at a position in the alignment.

• These degeneracies lead to complications in trying to find suitable annealing temperatures and degenerate primer lengths.

• The concentration of any single primer drops due to the degeneracy for accommodating more divergent genes. As a result, the number of primer molecules used for target amplification drops.

• Non-specific products are generated due to primers in the pool which do not participate in amplification of the targeted gene, but are available to prime non-specific synthesis.

• The use of short primers required for short conserved regions needs low stringency annealing conditions which exacerbate the above problems.

Seegene's unique Annealing Control Primer (ACP™) system, which, as a new generation technology to PCR, dramatically improves PCR specificity is particularly incorporated for amplifying most or all members of a gene family and was developed as a novel technology, SAFM™ (patent pending).

As a world first technology for amplifying most or all members of a gene family, SAFM™ Technology is specifically and initially applied to the isolation of a homeobox family and, easily and simply, enables researchers to screen and isolate most homeobox genes (>99%) from any vertebrates, including animals and humans, fungi, plants, fish, and insects.