Student worksheet demonstrating restriction enzymes and gel electrophoresis using colored pop-it beads to represent DNA fragments.

You're working on a hands-on activity simulating restriction enzyme digestion and gel electrophoresis using colored pop-it beads to represent DNA nucleotides. Let’s walk through the solution step by step.

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## 🔍 STEP 1: Understand the DNA Strand

The diagram shows a strand of 35 pop-it beads in this order (from 5’ → 3’):

> Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green, Blue, Red, Yellow, Green

Let’s number them for clarity (positions 1 to 35):

| Position | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35 |
|----------|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|
| Color | R | Y | G | B | R | Y | G | B | R | Y | G | B | R | Y | G | B | R | Y | G | B | R | Y | G | B | R | Y | G | B | R | Y | G | B | R | Y | G |

*(R = Red, Y = Yellow, G = Green, B = Blue)*

This is a repeating RYGB pattern — very helpful!

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## 🧬 STEP 2: Apply Each Enzyme Based on “Cuts Between”

We’ll go enzyme by enzyme.

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✔ ENZYME 1: Cuts Between Blue & Red (B-R)

Look for places where Blue is immediately followed by Red.

In our sequence:

- Position 4 (B) → 5 (R) → CUT
- Position 8 (B) → 9 (R) → CUT
- Position 12 (B) → 13 (R) → CUT
- Position 16 (B) → 17 (R) → CUT
- Position 20 (B) → 21 (R) → CUT
- Position 24 (B) → 25 (R) → CUT
- Position 28 (B) → 29 (R) → CUT
- Position 32 (B) → 33 (R) → CUT

That’s 8 cuts, which will produce 9 fragments.

Each fragment is 4 beads long (since pattern repeats every 4: RYGB).

✔ Fragment Sizes: All 9 fragments are size 4

➡️ In “Fragment Sizes” cell: Draw 9 groups of 4 beads each — e.g., `RYGB`, `RYGB`, ..., `RYGB`

➡️ In “Gel Banding Pattern”: Since all fragments are same size, you’ll see one thick band at position corresponding to size 4.

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✔ ENZYME 2: Cuts Between Yellow & Blue (Y-B)

Find positions where Yellow is followed by Blue.

Check:

- Position 2 (Y) → 3 (G) ✘
- Position 6 (Y) → 7 (G) ✘
- Position 10 (Y) → 11 (G) ✘
- ... Wait — actually, in our sequence, Yellow is always followed by Green, NOT Blue.

Wait — let’s double-check:

Sequence: R-Y-G-B-R-Y-G-B-...

So after Yellow comes Green → NO Y-B junctions!

✘ So Enzyme 2 makes NO cuts.

✔ Fragment Sizes: One fragment of size 35

➡️ Draw one long string of 35 beads: RYGBRYGB... up to 35.

➡️ Gel Banding Pattern: One band at size 35 (top of gel — largest fragment).

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✔ ENZYME 3: Cuts Between Red & Yellow OR Blue & Red (R-Y or B-R)

This enzyme cuts at two possible sites.

We already know from Enzyme 1 that B-R occurs 8 times.

Now check for R-Y:

- Position 1 (R) → 2 (Y) → CUT
- Position 5 (R) → 6 (Y) → CUT
- Position 9 (R) → 10 (Y) → CUT
- Position 13 (R) → 14 (Y) → CUT
- Position 17 (R) → 18 (Y) → CUT
- Position 21 (R) → 22 (Y) → CUT
- Position 25 (R) → 26 (Y) → CUT
- Position 29 (R) → 30 (Y) → CUT
- Position 33 (R) → 34 (Y) → CUT

That’s 9 R-Y cuts.

But wait — we also have 8 B-R cuts.

Are these overlapping? Let’s map all cut sites:

- R-Y cuts at positions: 1→2, 5→6, 9→10, 13→14, 17→18, 21→22, 25→26, 29→30, 33→34
- B-R cuts at positions: 4→5, 8→9, 12→13, 16→17, 20→21, 24→25, 28→29, 32→33

Note: Some positions are adjacent — for example, after position 4 (B), we cut before 5 (R) — and position 5 (R) is followed by 6 (Y), so we cut again between 5→6.

This means every 4 beads, there are two cuts: one at end of B (before R), and one at start of R (before Y). But since they’re adjacent, it creates fragments of size 1?

Wait — let’s think carefully.

Actually, cutting between B-R and R-Y means:

Between bead 4 (B) and 5 (R) → cut
Between bead 5 (R) and 6 (Y) → cut

So fragment between 4→5 is just bead 5? No — fragments are between cuts.

Let’s list all cut positions (between beads):

Cut positions (between bead n and n+1):

From R-Y: after bead 1, 5, 9, 13, 17, 21, 25, 29, 33
From B-R: after bead 4, 8, 12, 16, 20, 24, 28, 32

So total cut positions (sorted):

After bead: 1, 4, 5, 8, 9, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33

That’s 17 cut points → 18 fragments.

Now let’s compute fragment sizes:

- Bead 1 to cut after 1 → fragment: bead 1 → size 1 (R)
- Cut after 1 to cut after 4 → beads 2,3,4 → size 3 (Y,G,B)
- Cut after 4 to cut after 5 → bead 5 → size 1 (R)
- Cut after 5 to cut after 8 → beads 6,7,8 → size 3 (Y,G,B)
- Cut after 8 to cut after 9 → bead 9 → size 1 (R)
- ... and so on.

Pattern: Size 1 (R), Size 3 (YGB), Size 1 (R), Size 3 (YGB), ...

How many?

Total fragments: 18

Alternating: starts with size 1, ends with size 1 (since last cut is after 33, then beads 34–35 remain? Wait — let's check.

Total beads: 35

Cut after bead 33 → next fragment is beads 34–35 → that’s 2 beads: Y, G

Oh! We missed that.

Final fragment: after cut at 33 → beads 34 and 35 → Y and G → size 2

So full breakdown:

1. Bead 1 → R → size 1
2. Beads 2-4 → Y,G,B → size 3
3. Bead 5 → R → size 1
4. Beads 6-8 → Y,G,B → size 3
5. Bead 9 → R → size 1
6. Beads 10-12 → Y,G,B → size 3
7. Bead 13 → R → size 1
8. Beads 14-16 → Y,G,B → size 3
9. Bead 17 → R → size 1
10. Beads 18-20 → Y,G,B → size 3
11. Bead 21 → R → size 1
12. Beads 22-24 → Y,G,B → size 3
13. Bead 25 → R → size 1
14. Beads 26-28 → Y,G,B → size 3
15. Bead 29 → R → size 1
16. Beads 30-32 → Y,G,B → size 3
17. Bead 33 → R → size 1
18. Beads 34-35 → Y,G → size 2 ← FINAL FRAGMENT

✔ So fragment sizes:
Nine fragments of size 1 (R)
Eight fragments of size 3 (YGB)
One fragment of size 2 (YG)

➡️ In “Fragment Sizes”: Draw 9 single red beads, 8 triplets (Y-G-B), and 1 pair (Y-G)

➡️ In “Gel Banding Pattern”: You’ll see three bands:
- One at size 1 (smallest, bottom)
- One at size 2 (middle)
- One at size 3 (top among small ones)

(Note: in real gels, smaller fragments travel farther — so size 1 at bottom, size 3 near top)

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✔ ENZYME 4: Cuts Between Green & Yellow (G-Y)

Look for G followed by Y.

In our sequence: R-Y-G-B-R-Y-G-B...

After G is always B → NO G-Y junctions

✘ So Enzyme 4 makes NO cuts

✔ Fragment: one fragment of size 35

➡️ Same as Enzyme 2: draw full 35-bead strand

➡️ Gel: one band at top (size 35)

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✔ ENZYMES 2 & 3: First Enzyme 2, then Enzyme 3

Remember: Enzyme 2 makes no cuts → so the DNA remains one piece of 35 beads.

Then Enzyme 3 cuts that 35-bead strand at its sites: R-Y or B-R.

We already did this above for Enzyme 3 alone — and got 18 fragments.

Since Enzyme 2 didn’t cut anything, applying Enzyme 3 afterward gives the same result as Enzyme 3 alone.

✔ So same as Enzyme 3:
- 9 fragments of size 1 (R)
- 8 fragments of size 3 (YGB)
- 1 fragment of size 2 (YG)

➡️ Fragment Sizes: same as Enzyme 3

➡️ Gel Banding Pattern: same as Enzyme 3 — three bands at sizes 1, 2, 3

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## 📊 Final Table Summary

Here’s how to fill your table:

| Enzyme | Cuts Between | Fragment Sizes | Gel Banding Pattern |
|---------------|--------------------|--------------------------------------------------------------------------------|------------------------------------------------|
| Enzyme 1 | Blue & Red | 9 fragments of size 4 (each RYGB) | One band at size 4 |
| Enzyme 2 | Yellow & Blue | 1 fragment of size 35 | One band at size 35 (top) |
| Enzyme 3 | Red & Yellow OR Blue & Red | 9× size 1 (R), 8× size 3 (YGB), 1× size 2 (YG) | Three bands: size 1 (bottom), 2 (middle), 3 (top) |
| Enzyme 4 | Green & Yellow | 1 fragment of size 35 | One band at size 35 (top) |
| Enzymes 2&3 | (Enzyme 2 first, then 3) | Same as Enzyme 3: 9×1, 8×3, 1×2 | Same as Enzyme 3 |

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## 🖍️ How to Draw in Your Worksheet

Use colored pencils to match the bead colors:

- Red = red pencil
- Yellow = yellow pencil
- Green = green pencil
- Blue = blue pencil

For “Fragment Sizes” column:

- Draw short lines or boxes with colored dots representing each fragment.
- E.g., for Enzyme 1: draw 9 groups like `[R][Y][G][B]`
- For Enzyme 3: draw 9 single `[R]`, 8 `[Y][G][B]`, and 1 `[Y][G]`

For “Gel Banding Pattern”:

- Draw a vertical rectangle (the gel).
- Draw horizontal lines (bands) at different heights:
- Top = large fragments (size 35)
- Bottom = small fragments (size 1)
- Middle = medium (size 2, 3, 4)

Label bands with their sizes if needed.

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## 💡 Key Concepts Reinforced

- Restriction enzymes cut DNA at specific sequences (here simulated by color pairs).
- Gel electrophoresis separates DNA fragments by size — smaller fragments move farther.
- Multiple enzymes can be used sequentially to create complex fragment patterns.
- Even if an enzyme doesn’t cut initially (like Enzyme 2), it may affect later digestion if it alters the DNA — but here, since no cut, no effect.

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✔ You’ve now solved the entire activity! Great job simulating molecular biology with pop-it beads. This is a fantastic way to visualize restriction digestion and gel separation.