PCR+of+Target+Gene!

Home Editors: Jordan Dickstein & Brian Hsia

**__What is PCR?__**
PCR is a common lab technique used to amplify a specific DNA sequence; in other words, PCR allows us to make many replications of a piece of DNA. Twenty-one cycles of PCR can produce over one million copies of a DNA sequence. PCR occurs in a thermal cycler, in which we put tubes filled with Taq polymerase, magnesium ions, free floating nucleotides, primers, and the DNA strand with the targeted DNA sequence. Taq polymerase is the enzyme responsible for DNA replication. The free floating nucleotides will be strung together by the Taq polymerase to create the new strands. Primers are short DNA segments that vary from 15-24 base pairs in length; they are complimentary to parts of the original DNA strand so that the gene of interest (the segment to be amplified) is on the 3' end of the primer. This arrangement is crucial because all DNA strands are built in the 5' to 3' direction. Having the gene of interest on the 3' end ensures that it is included in amplification.

Below delineates the general process of PCR. The arrows designate the direction that a DNA strand is being created. The primers are complimentary to the strand so that the gene of interest that needs to be amplified is in the middle. (Note: the area that the PCR is complimentary to is included in the amplification.) The TAQ polymerase is responsible for the creation of the new DNA strands. Inside the thermal cycler, there are cycles of heating and cooling that occur to facilitate this process. There is an initial heating to separate the two original DNA strands in the helix; this step is called the denaturation step. Annealing of the primers follows, involving a cooling in the machine to allow the primers to attach to their respective locations on the separated DNA strands. Finally, there is the elongation step, heating the tubes again to allow the Taq polymerase to attach to the primers and create a new DNA strand in the 5' to 3' direction. This process of heating, cooling, and heating is just one cycle of PCR. Overall, PCR is very effective in creating many copies of a segment of DNA. The number of copies made per strand is an exponential progression with respect to the number of cycles of PCR. (There are 2^x total strands created from each helix of DNA, with x being the number of PCR cycles.)

PCR of Target Gene in Relation to our RNAi Project:
__The Big Picture:__ Our overall goal for this project is to make changes in gene expression so that the C. elegens worm expresse the Rol-6 gene. By inserting double-stranded RNA, in the recombinant plasmid we can destroy the mRNA of our targeted gene inside the worm. DNA is transcribed into mRNA, which codes for proteins and gene expression. This process of creating dsRNA would prevent the creation of the protein involved in movement of the worm. In order to get this dsRNA into the cells, the target gene must flanked by the T7 promoters on both sides so when the gene is transcribed repeatedly, the gene of interest will be expressed.

__Our Focus:__ We need to be able to amplify a gene in the genomic DNA of //C.elegans// so that this gene can be inserted into the vector L4440, creating a recombinant plasmid (L4440 with the gene insert). This process allows us to create many copies of this gene so that when we digest and ligate the gene and the vector, there will be a high probability that the vector will include the insert during ligation. PCR ensures high probability of successful recombinance, allowing us to insert the targeted gene between the two T7 promoters on L4440, thereby allowing the worms that eventually take in this plasmid to produce dsRNA. Our step is crucial in preventing the production of the protein coded for by our target gene, Rol-6. Before we perform PCR, we needed to design the primers and restriction sites in order to correctly amplify the gene and prepare for ligation it into the vector that will eventually transform the cells.

**Designing the Primers**
Gene of choice: Rol-6

Objective: To create primers to amplify a segment of DNA in the Rol-6 gene. At the same time, we must engineer in specific restriction sites into the primers so that the amplified piece of DNA may be ligated into the L4440 vector. We were wary of the following guidelines when designing the primers:
 * The amplified piece must be small enough to make the L4440 vector small to increase the chances of transformation, but it must also be large enough to analyze on an electrophoresis gel and be transcribed to create dsRNA.
 * The restriction sites in the primers must exist in the multiple cloning site between the T7 promoters in L4440, so that after successful insertion of the amplified piece, RNA polymerase will bind to both T7 promoters and transcribe the insert from both directions, creating dsRNA.
 * The restriction sites engineered into each primer must be different from each other to minimize ligation of the original L4440 pieces and maximize insertion of the Rol-6 gene.
 * The forward and reverse primers must be complimentary to opposing strands of the targeted gene and must exist on either side of the targeted sequence so that the Taq polymerase builds the new strand from 5' to 3' towards the targeted sequence during PCR.
 * The primers should have melting temperatures within one degree Celsius of each other.
 * The restriction enzymes chosen should be compatible in a double digest (matching in NEB buffers and incubating temperatures).

Procedure:
 * 1) We chose the following restriction enzymes to use, SacII and HindIII, which have restriction sites between the T7 promoters in L4440. They are both compatible in double digest; we will use NEB buffer #2 and incubate at 37 degrees Celsius.
 * 2) Choosing a sequence in Rol-6 to amplify, found on wormbase.org, we entered the sequence into idtDNA.com to create the forward and reverse primers and entered the restriction site sequences for SacII and HindIII on the 5' ends of the primers. The restriction sites do not need to be complimentary to the DNA strand.
 * 3) To adjust the melting temperatures of the primers, we added nucleotide tails onto the restriction sites of the primers - a string of guanine or cytosine bases to significantly raise the melting temperature or a string of adenine or thymine bases to raise the melting temperature only slightly.
 * 4) After ordering the primers, we will run PCR with the isolated genomic DNA from //C.elegans,// primers, and master mix to amplify the targeted sequence for digestion and ligation into the L4440 vector.

PCR Verification: We ran this verification electrophoresis gel to ensure that we had the correct amplification.

__PCR Confirmation Gel Using 100 bp Ladder__

Final Product
 Gene of choice: Rol-6 (Note: All sequences are written 5' to 3') **Targeted Amplification ** within Gene Sequence (247 bp):  **accaaagttcccaggaggtggattcccagatggtccattcccgaatggaggag gaccacgtggaggaaatcaatgtcaatgcactgttgagaactcatgcccaccaggaccagctggaccagaaggagaggaa ggaccagatgggcacgatggacaagacggagtcccaggatttgacggaaaagatgccgaagatgttcaaaacaccccacc aacaggatgcttcacctgtccacaaggaccactt **

**Forward primer **  containing **HindIII **  restriction site: **__GGG G__ ** **<span style="font-family: Arial,Helvetica,sans-serif;">AA GCT T <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">AC ** <span style="font-family: monospace,'Sans Serif',Arial; line-height: normal;">**<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;"> CAA AGT TCC CAG GAG GTG GAT T ** <span style="font-family: Arial,Helvetica,sans-serif;"> HindIII sequence: AAGCTT

<span style="background-color: #008080; font-family: monospace,'Sans Serif',Arial; line-height: normal;">**<span style="font-family: Arial,Helvetica,sans-serif;">Reverse primer ** <span style="font-family: Arial,Helvetica,sans-serif;"> containing <span style="background-color: #00ff00; font-family: monospace,'Sans Serif',Arial; line-height: normal;">**<span style="font-family: Arial,Helvetica,sans-serif;">SacII ** <span style="font-family: Arial,Helvetica,sans-serif; line-height: normal;"> restriction site: <span style="font-family: monospace,'Sans Serif',Arial; line-height: normal;">**<span style="font-family: Arial,Helvetica,sans-serif;">A ****<span style="font-family: Arial,Helvetica,sans-serif;">AA A <span style="background-color: #00ff00; font-family: Arial,Helvetica,sans-serif;">CC GCG G <span style="font-family: Arial,Helvetica,sans-serif;">AA GTG GTC CTT GTG GAC AGG TGA A ** SacII sequence: CCGCGG

The following section highlighted in red is where the forward primer is complimentary to on the opposing strand. The section highlighted in blue is where the reverse primer is complementary. <span style="font-family: monospace,'Sans Serif',Arial; line-height: normal;">**<span style="background-color: #ff0000; font-family: Arial,Helvetica,sans-serif;">accaaagttcccaggaggtggatt <span style="font-family: Arial,Helvetica,sans-serif;">cccagatggtccattcccgaatggaggag gaccacgtggaggaaatcaatgtcaatgcactgttgagaactcatgcccaccaggaccagctggaccagaaggagaggaa ggaccagatgggcacgatggacaagacggagtcccaggatttgacggaaaagatgccgaagatgttcaaaacaccccacc aacaggatgc <span style="background-color: #00ffff; font-family: Arial,Helvetica,sans-serif;">ttcacctgtccacaaggaccactt **

After PCR, the resulting product, including the added primers and restriction sites, will be 267 bp long.