Overlap-extension PCR: a promoter, a gene and a tag

• You will end with: a 769 bp linear fusion of a promoter, a gene and a tag (160 + 540 + 69 bp), the gene reading straight into the tag.
• Time: about 20 minutes.
• Files: OE-Promoter.gb, OE-Gene.gb, OE-Tag.gb, OE-bad-Tag.gb (exercise 2 only).
• Before this: Gibson; Before you start if the Bench or the sample files are new to you.

Before you click anything, close every tab you do not need: the panel lets you choose any open tab.

Exercise 1 of 2: three fragments, one fusion

1. Choose File ▸ Open and select OE-Tag.gb, OE-Promoter.gb and OE-Gene.gb (or drag them onto the window). The tag is deliberately opened first.

   If instead you see other tabs, close them: the panel would offer them as fragments.

2. Click the Bench tab.
3. Click Overlap PCR at the top of the middle column.

   You see the heading Overlap PCR, with fuse by PCR, tails that overlap beside it. Until you choose fragments it says Choose a tab, and the part of it to use, for every fragment. Open the pieces you want to fuse first.

4. Under Fragments to fuse, set From of fragment 1 to OE-Promoter.

   You see the Part beside it read synthetic promoter (promoter), 41–200, 160 bp.

5. Set From of fragment 2 to OE-Gene.

   You see its Part read synthGene (CDS), 51–590, 540 bp.

   Why

   A fragment is a tab and a part of it, here a feature. The padding outside the feature stays out of the fusion; the other choice in the list, All of it, would take it in.

6. Click Add fragment.
7. Set From of fragment 3 to OE-Tag.

   You see its Part read synthTag (CDS), 41–109, 69 bp.

8. Leave Overlap Tm at 60 °C (default).
9. Compare the six primers below the fragment list with this table, in the order to ask for them:

   Primer	What the panel says
   F1	24 nt, forward, outer at 60 °C. No tail.
   R1	49 nt, reverse: 19 of the neighbour, 30 annealing at 57 °C
   F2	46 nt, forward: 19 of the neighbour, 27 annealing at 61 °C
   R2	37 nt, reverse: 10 of the neighbour, 27 annealing at 60 °C
   F3	28 nt, forward: 10 of the neighbour, 18 annealing at 67 °C
   R3	21 nt, reverse, outer at 61 °C

   Each name starts with the fragments' tab names (SOE OE-Promoter-OE-Gene-OE-Tag and then F1, R1 and so on) until you name the product in step 11. The sequence under each carries the tail in upper case and the annealing part in lower case: R1 begins CCTCCAAATGATCTATCAT in upper case, the reverse complement of the gene's first 19 bases, then aggcttctaag and on.

   Why

   R1 and F2 are the two inner primers of the first junction. R1 belongs to the promoter and ends in the start of the gene; F2 belongs to the gene and starts with the end of the promoter. Their tails are complementary to each other, which is the whole mechanism. The primers differ in length and Tm because each annealing part grows to about 60 °C within 18 and 30 bases; F3 (18 annealing at 67 °C) is the shortest the app makes, on a GC-rich start.

10. Compare the lines under the primers with this table:

    Line	Between
    Junction 1: 38 bp overlap, Tm 61 °C, with the shared bases	promoter and gene
    Junction 2: 20 bp overlap, Tm 61 °C	gene and tag
    First round: 179 bp, 569 bp, 79 bp. Fused: 769 bp.
    Product: 769 bp, linear · synthetic promoter, synthGene, synthTag

    No warning appears under the product.

    If instead you see a warning under the product, compare the three Part lines with steps 4, 5 and 7.

11. Type Promoter-Gene-Tag in Name of the fused product.

    You see the primers renamed Promoter-Gene-Tag F1 to Promoter-Gene-Tag R3; the product will take the same name.

12. Optional: click Save primers.

    You see Saved 6 primers to My primers.

13. Set Overlap Tm to 65 °C.

    You see junction 1 become 40 bp overlap, Tm 62 °C (40 is the longest the app will make) and junction 2 23 bp overlap, Tm 65 °C; the first round reads 180 bp, 571 bp, 81 bp.

14. Set Overlap Tm to 52 °C.

    You see 28 bp, Tm 52 °C and 18 bp, Tm 57 °C; the first round reads 174 bp, 563 bp, 78 bp. Fused stays 769 bp at either setting.

15. Set Overlap Tm back to 60 °C (default).
16. Click Open product.

    You see a new tab, Promoter-Gene-Tag, with the Features tab in front, listing synthetic promoter, synthGene and synthTag, each once, and the primers drawn on the molecule.

    • The new tab is 769 bp, linear.

    If not, go back to the panel and check the three Part choices.

    Why

    The first-round sizes add up to more than the product: 179 + 569 + 79 = 827, but the product is 769. The difference, 58 bp, is the two overlaps (38 + 20), each shared by two products and present once in the fusion. And synthGene and synthTag are drawn a little short, from base 180 rather than 161 and from 711 rather than 701: the part of each that lies in an overlap came from its neighbour's first round as a tail, so the annotation starts after it. The sequence is whole.

    The product is a scratch document with how it was made recorded: File ▸ Save if you want to keep it. Protocol… under the map lists the six primers to order and the fusion as a reaction of its own.

17. Open the product's ORFs tab.

    You see one reading frame, forward, bases 161 to 769, 202 amino acids (180 of the gene and 22 of the tag) and then the stop.

Exercise 2 of 2: a tag that is a base off

OE-bad-Tag.gb is OE-Tag with one extra base in front of its start codon. Its synthTag feature starts on that base and is marked /codon_start=2 (it reads from its second base), which is what you would have if a linker you designed by hand were one base too long. Nothing about the overlap is wrong: the pieces fuse perfectly. What goes wrong is the frame.

1. Choose File ▸ Open and select OE-bad-Tag.gb. Leave the others open.
2. Click the Bench tab.
3. Click Overlap PCR.
4. Set From of fragment 3 to OE-bad-Tag.

   You see its Part read synthTag (CDS), 41–110, 70 bp, and the product now 770 bp, linear.

   You also see a warning under the product, The parts assemble, but the tube may also give something else: and then synthGene runs into synthTag at junction 2 out of frame: the fusion shifts the reading frame of synthTag.

   Why

   The panel checks, at each junction, the frame of each gene on either side, and says where they disagree. The fusion would be made, and then the tag would be read in the wrong frame.

5. Optional: click Open product, then open its ORFs tab.

   You see nothing of 75 codons or more.

   Why

   The reading that starts at the gene's ATG does not meet a stop where the tag should end it: the stop is now out of frame, and the molecule runs out first. In the lab you would have found a protein with no tag.

6. Back in the panel, set From of fragment 3 back to OE-Tag.

   You see the warning gone.

Done when:

• The exercise 1 product is 769 bp, linear, with synthetic promoter, synthGene and synthTag each once.
• The warning about junction 2 is gone once fragment 3 is OE-Tag again.

How it works

The five words of this page:

• Overlap: a stretch that the end of one fragment shares with the start of the next. After the first round of PCR the two products carry it, and it is what lets them find each other. The app grows it until it melts at a chosen temperature, taking half from each neighbour.
• Tail: the 5′ end of a primer that does not pair with its own template. An inner primer's tail is a copy of its neighbour's end, in upper case in the panel.
• Annealing part: the 3′ end of the primer that does pair with the template and starts the copying. The app grows it until it melts at about 60 °C.
• Outer and inner primers: the two ends of the fusion are amplified by outer primers, which have no tail. Every junction in between gets two inner primers, one for each side, complementary to each other over the whole overlap.
• First-round products: what each fragment's primer pair makes from its own template. Each is a little longer than its fragment, by the tails.

Each file is a short linear template with the part you want inside it and some flanking sequence you do not. All four are synthetic examples (invented sequence).

synthGene is an open reading frame of 180 codons that starts on an ATG and has no stop codon, so a tag can follow it. synthTag is 23 codons: a start codon, a glycine-serine linker, six histidines and a stop. The goal is one molecule, promoter then gene then tag, 160 + 540 + 69 = 769 bp, in which the gene reads straight into the tag.

In the lab this is three PCRs and a fusion. Each fragment is amplified from its own template with its own primer pair (F1 and R1 on the promoter, F2 and R2 on the gene, F3 and R3 on the tag). The three products are mixed with no primers and cycled, so they anneal at their overlaps and extend into one molecule. Then the two outer primers, F1 and R3, amplify that. The panel does all of it on the screen, with the same PCR simulation as the PCR panel, and gives you the primers to order.

The fragments are fused in the order of the list; the arrows beside each one move it, and Remove drops it (two stay at least). The tabs may be opened in any order, because you choose the order of the fragments in the panel. This is the reaction for fragments with no site and no vector, only the tails of the primers.

What the exercise 1 product holds, beyond what the steps showed:

• Each overlap (38 and 20 bp) appears once in the product, and the three sequences it was made from are all there, in that order.
• The promoter ends at base 160 and ATG is bases 161 to 163: the gene starts exactly where the promoter stops.
• Translated, the reading frame begins MIDHLEG and ends ...MGSGSGSHHHHHHGSARARTRS*: gene, linker, six histidines, stop, in one frame.
• A fusion is a straight piece of DNA, so unlike the Gibson tutorial it does not close into a circle.
• The Enzymes tab has the default ticks, every enzyme that cuts the product once. An overlap PCR has no enzyme of its own to tick.

The overlap decides how well the first-round products hold on to each other and nothing else: a longer one is a more robust fusion and a longer primer to order, but the molecule is the same. Both overlaps melt well over 50 °C and are over 15 bases, the limits under which the panel warns. A deletion made with two pieces of the same tab gets a warning of the same kind as in exercise 2 when what is left out is not a multiple of three bases. As there, the fix is in the design (the bases between the two parts), not in a setting of the panel.

Next

Overlap PCR on the Bench, including the warnings it adds (a short or non-unique overlap, a primer that melts well below its target) and how a deletion is made with the same tab twice. The primers are the ones the PCR panel simulates; for the reaction that joins pieces with an enzyme mix instead, see Gibson. Back to the tutorials index.