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RAM - Ramification Amplication Method

Introduction to RAM

With our RAM technology, a billion copies of a gene sequence can quickly and accurately be created (super-exponential amplification), in turn increasing discrimination power up to 1000 fold above current methods. Unlike other amplification techniques that require multiple cycles of alternating temperatures, RAM is an isothermal process that operates at a single temperature of 30°C.

In actual clinical conditions, RAM is able to detect fewer than 5 targets and to produce one billion copies of that target in 1 hour*. This method is also used with extreme simplicity and sensitivity to detect viruses and bacteria in clinical specimens. In-situ (on a slide) detection of multiple viral sequences in cells is also possible using RAM technology.

The Components

The components required from RAM amplification include: 1) presence of a target nucleic acid sequence; 2) a circularizable probe; 3) a ligase; 4) DNA poylmerase; and 5) two extension primers, Primer 1 and Primer 2.

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How RAM Works

Figure 1

Figure 2

When the target nucleotide sequence is present, the 5' and 3' ends of the circularizable probe attach to the target nucleic acid in a way that an open loop is formed (see Figures 1 & 2).
Figure 3

Before amplification begins, the loop must be "closed." This closure is accomplished through the use of a ligase that links the ends of the primer together and forms a closed, circular amplification probe (Figure 3). The resultant circular probe is a single stranded DNA, of which its nucleic acids are not paired to those on another strand of DNA. This single stranded DNA is used as a template for the creation of complementary strands of DNA.

Figure 4

The first extension primer, Primer 1, which is complementary to a portion of the circular amplification probe, attaches to the circular probe (Figure 4).

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Figure 5 At this point the DNA polymerase comes into play. DNA polymerase is an enzyme that catalyzes the synthesis of complementary base pairs along the single stranded nucleic acid template. The DNA polymerase starts at the extension primer and begins creating new nucleic acids to pair with those on the template, a process called strand extension (Figure 5).
Figure 6 This extension also causes the initial target nucleic acid strand to be separated from the circular probe (Figure 6).
Figure 7

The strand extension continues along the circumference of the circle and results in the formation of a double stranded DNA (Figure 7).

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When the starting point is reached (e.g. one complete rotation around the circular probe), the newly formed Primer 1 strand is displaced incrementally and the DNA polymerase makes another trip around the circular probe. For each rotation around the circular probe, the displaced Primer 1 strand gets longer. (See Figure 8 below.)

The newly displaced Primer 1 linear strand also acts as DNA a template. The second extension primer, Primer 2, which is complementary to a portion of this linear DNA template, binds to this linear DNA template. Once again, the DNA polymerase creates new nucleic acids along the template.

As each rotation around the circular probe is completed, the linear DNA template gets longer allowing another Primer 2 to attach and extend. As the new Primer 2 linear strands extend, they eventually displace the "downstream" strands. This results in branching and creation of yet another set of linear DNA templates, to which the Primer 1 is complementary. Thus, Primer 1 attaches to the displaced Primer 2 linear strands and the process of extension, displacement and branching continues. (See Figures 9 & 10 below.)

The end result of this amplification process is a super-exponential multiplication of the initial target sequence, which produces over a billion target sequences for easy detection.

Figure 8 Figure 9 Figure 10
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Figures 8, 9, 10

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