Introduction

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by progressive degeneration of anterior horn cells in the spinal cord. In ~94% of SMA cases, the molecular basis is a homozygous deletion of exon 7 in the SMN1 gene.

Detecting this specific deletion is crucial for accurate diagnosis and genetic counseling. Among the earliest and most elegant tools developed for this purpose is the PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay using the DraI restriction enzyme. This article explores the science and strategy behind this method.

The Biological Basis: SMN1 vs SMN2

The SMN1 gene is essential for motor neuron survival. Its near-identical paralog, SMN2, differs by only a few nucleotides but is functionally less effective due to altered splicing.

The critical single nucleotide difference lies at c.840 in exon 7:

Although silent at the amino acid level, this base difference is pivotal in diagnostic assays.

Principle of the DraI-based PCR-RFLP Assay

The assay hinges on selective digestion of SMN2 exon 7 PCR products by the DraI enzyme, while SMN1 products remain undigested. This is achieved using a cleverly designed reverse primer that introduces a DraI restriction site only when amplifying SMN2.

We can’t do an isolated PCR to find out the presence of exon 7 in SMN1 to diagnose SMA, as exon 7 of SMN2 will also be amplified due to sequence homology.

So our goal is to make a restriction enzyme site (DraI) appear only in the SMN2 PCR product. This will make sure that, after digestion with DraI, only SMN2 gets cut, not SMN1. DraI is a restriction enzyme isolated from Deinococcus radiodurans that recognizes the palindromic DNA sequence TTT^AAA and cuts between T and A.

We do this by intentionally design the reverse primer in a way that it overlaps the 840 position and changes one or more bases (called a primer mismatch) to artificially create the DraI recognition site only when the template is SMN2.

To create a DraI site (TTTAAA) in the SMN2 PCR product, we must ensure the sequence contains:

TTTAAA

What does SMN2 naturally have?

Let’s look again at the native sequence of SMN2 exon 7 (top strand):

c.838  c.839  c.840  c.841  c.842  c.843  c.844
   T      T      T      T      C      A      A
       (DraI site starts at 838)

So:

• c.838–c.840 = TTT ✅
• c.842–c.844 = CAA. We need to convert to AAA for recognition of DraI.

To convert CAA → AAA, we must change the C at c.842 to A.

How do we do that?

We design the reverse primer (which binds to the bottom strand) such that:

• It deliberately mismatches the base at c.842.
• The normal complementary base to C (on top strand) is G (on bottom strand).
• So, we replace that G with a T in the reverse primer, so the polymerase writes A instead of C.

Great — now let’s look at SMN1, step by step, just like we did for SMN2, and understand why the DraI site (TTTAAA) is not formed in SMN1, even after PCR using the same mismatch primer.

Why does SMN1 remain unaffected?

The key difference is at c.840:

• SMN2 has T → helps form TTT at c.838–c.840 which is a part of DraI site
• SMN1 has C at c.840 → so the sequence becomes TTC at c.838–c.840 which is not part of the DraI site

Let’s write the top strand sequence of SMN1 exon 7 (from c.838 to c.844):

Position:    c.838  c.839  c.840  c.841  c.842  c.843  c.844
Top strand:     T      T      C      T      C      A      A

Now break this into the two parts we need for DraI:

• First half of DraI site (expected TTT) = TTC (not a part of DraI recognition site)
• Second half (we will modify to AAA using primer) = we try to convert CAA (at c.842–844) to AAA ✅

So with the mismatch primer, we can successfully convert:

• CAA → AAA (by changing C at c.842 to A, as before)

But the problem is:

• The first three bases (c.838–c.840) are TTC, not TTT
  → So the full sequence becomes: TTCAAA, not TTTAAA

Methodology

1. PCR Amplification

A segment spanning exon 7 is amplified using:

• A common forward primer
• A modified reverse primer designed to:
  • Bind to both SMN1 and SMN2
  • Introduce a TTTAAA DraI site in SMN2 products only

This is possible because SMN2 has a T at position 840, allowing perfect pairing. In SMN1 (with a C → G on the template strand), this base mismatch disrupts the DraI site.

2. DraI Digestion

• DraI is a restriction enzyme that recognizes and cuts at the site: 5′ — TTT↓AAA — 3′
• After PCR, the products are incubated with DraI:
  • SMN2 products get cut
  • SMN1 products remain uncut

3. Gel Electrophoresis

The digested products are run on an agarose or polyacrylamide gel:

This banding pattern helps distinguish normal individuals, carriers, and affected patients.

Advantages of the Method

• Cost-effective and rapid
• Distinguishes SMN1 from SMN2 without sequencing
• Still used in many settings with limited access to real-time PCR or MLPA

Limitations

• Cannot detect carriers (with one copy of SMN1) quantitatively
• May miss small intragenic mutations
• Cannot find out SMN2 copy numbers

Conclusion

The DraI-based PCR-RFLP method remains a brilliant example of how a single nucleotide difference, when coupled with intelligent primer design, can form the basis of a robust diagnostic assay. While newer technologies provide greater depth, this method continues to have a valuable place in the clinical genetics toolkit — especially in resource-limited settings.

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