Overlap Extension PCR

Assembling Two Fragments Using Overlap PCR

September 25th, 2022

Overview

As discussed in last week's blog, fragments taken from Deinococcus radiodurans' DNA, namely the left fragment and right fragment of the fbaA gene, and one taken from the Escherichia coli's plasmid, namely the kanamycin resistance gene, are the fragments which were intended to be assembled using Overlap PCR. To extract the fragments, the appropriate primers were used in a traditional PCR using template DNA from both D. radiodurans and E. coli. The traditional PCR procedure utilizes the Q5 High Fidelity PCR protocol using the Q5 buffer and polymerase. After amplifications, the fragments were run on a gel to verify the bands were of the correct size including overhangs; the left fragment being 582 bp, the kanamycin resistance fragment 947 bp, and the right fragment 657 bp. Due to past gel extraction procedures resulting in a low DNA yield, the bands were not gel extracted, so the Overlap PCR was attempted with the direct products of the traditional PCR. It was hypothesized that matching the primers' annealing temperature during both part of the Overlap PCR would result in the assembly of each of the two fragments, one being left plus kanamycin resistance and the other being right plus kanamycin resistance. 

Procedure

Before beginning the Overlap PCR procedure, the appropriate volumes of each fragment that go into the PCR tube were calculated using the following steps:

Since a 100 ng/ul DNA is needed per reaction, 100 was divided by the fluorometer concentration of each fragment which was then divided by the size of each fragment as such:

Left fragment: (100 ng) / (26.3 ng/ul) = 3.80 ul / 0.582 kb = 6.53 ul

Kanamycin resistance: (100 ng) / (22.2 ng/ul) = 4.50 ul / 0.947 kb = 4.76 ul

Right fragment: (100 ng) / (17.1 ng/ul) = 5.85 ul / 0.657 kb = 8.90 ul

Then, during step 1 of the Overlap PCR, two PCR tubes were labeled left+kan and right+kan. In each tube there is a total volume of 50 ul:

22.5 ul overlap PCR master mix

Tube 1: 6.53 ul left + 4.76 ul kan

Tube 2: 8.90 ul right + 4.76 ul kan

PCR water is added to both tubes up to the 50 ul limit

(notice, during step 1 of the overlap PCR, no primers are added because the intention is to assemble the overhang of one fragment with the homologous overhang region of the other fragment by overlapping)

Step 1 Overlap PCR conditions were as follows: (* denotes the degree symbol)

Lid: 105 *C

Volume: 50 ul

1) 94 *C, 0:30 sec

2) 94 *C, 0:15 sec

3) 66.5 *C, 0:30 sec

-0.5 *C per cycle 

4) GOTO step 2, 9X

5) 94 *C, 0:15 sec

6) 62 *C, 0:15 sec

-0.5 *C per cycle

7) 72 *C, 0:30 sec

8) GOTO step 5, 5X

9) 72 *C, 2:00 min

10) Hold: 4 *C, infinity

Step 1 of the Overlap PCR takes about 30 minutes in the Thermocycler. After the Overlap PCR run is complete, the products are taken out of the Thermocycler to use for step 2. 

During step 2 of the Overlap PCR procedure, two tubes are labeled similarly to step 1 tubes. In each tube there is a total volume of 50 ul:

22.5 ul overlap PCR master mix

Tube 1: 4 ul PCR products from step 1 tube 1 + 1 ul forward primer (1) and 1 ul reverse primer (4)

Tube 2: 4 ul PCR products from step 1 tube 2 + 1 ul forward primer (3) and 1 ul reverse primer (6)

PCR water is added to both tubes up to the 50 ul limit

(notice , during step 2 of the Overlap PCR, the primers are added to amplify the fragments that should have overlapped in step 1)

Step 2 Overlap PCR conditions were as follows: (* denotes the degree symbol)

Lid: 105 *C

Volume: 50 ul

1) 94 *C, 0:30 sec

2) 94 *C, 0:15 sec

3) 69 *C, 0:15 sec

-0.5 *C per cycle 

4) 72 *C, 0:30 sec

5) GOTO step 2, 17X

6) 94 *C, 0:15 sec

7) 61 *C, 0:15 sec

8) 72 *C, 0:30 sec

9) GOTO step 6, 23X

10) 72 *C, 2:00 min

11) Hold: 4 *C, infinity

Step 2 of the Overlap PCR takes about 1.5 hours in the Thermocycler. 

Results

After both steps of the Overlap PCR are completed, the fluorometer concentration of the potentially assembled fragments, left+kan and right+kan, is taken.

Left+kan = 20.6 ng/ul

Right+kan  = 26.2 ng/ul

With this in mind, the products were run on a 2% agarose gel using 20 ul of the above PCR products due to the low concentration. Both 100  bp and 1 kb ladders were also used. The resulting gel electrophoresis looked like the following: (the order in which the products were loaded is left+kan in the 3rd well and right+kan in the 5th well)

As seen above, no distinct bands of a certain size were present. Instead, a smear existed for both left+kan and right+kan. This signifies that the Overlap PCR did not result in the appropriate assembly of the desired fragments. The hypothesis that using the primers' annealing temperature during both steps of the Overlap PCR is nullified. Because no primers were added during step 1 of the procedure, the annealing temperature should have matched the optimal temperature of the overhang regions, not the primers. In the future, the Overlap PCR will be reattempted with a tweak to the PCR conditions to account for the overhang regions' optimal annealing temperature. As a final note, the three individual fragments will be reamplified from D. radiodurans and E. coli. plasmid DNA using traditional PCR to insure the best results.