The+Replication+of+Our+Isolated+Bli-1

=// PCR // media type="youtube" key="x5yPkxCLads" height="405" width="660" = =// Relevance //= In order to induce RNAi in the //C. elegans//, a number of procedures must be done that utilize a process called PCR. After extracting the DNA from the worms, PCR is utilized to amplify the target sequence. This amplified product is then ligated into the vector of L4440, which is transformed next into the DH5-alpha to be produced by the cells in high quantities. The vectors with inserts eventually are transformed into the HT115(DE3) cells and fed to the //C. elegans//, which will lead to RNAi of the blister-1 gene. See below for our results from the PCR amplification attempts. Our need for amplification was successfully fulfilled by using PCR in this lab, and eventually PCR worked successfully (though not always on the first attempt).

=// Brief History //=

PCR, acronym for Polymerase Chain Reaction, was invented by Nobel Prize winning chemist Dr. Kary Banks Mullis, who was also awarded the prestigious Japan Prize in 1993 for this award winning discovery. His introduction of the PCR process revolutionized the scientific world in 1993. Currently, Dr. Mullis is a Distinguished Researcher at the Children's Hospital and Research Institute in Oakland, while still working on multiple companies' boards of advisors, giving his insight on DNA legal matters, and lecturing at college campuses, corporations and academic meetings worldwide. To read Dr. Kary Banks Mullis's biography, click here.

= // The Process // = media type="youtube" key="ZmqqRPISg0g" height="525" width="660" =// The Active Players //= __**//Taq// polymerase**__- abbreviation for Thermus Aquaticus, a thermophilic bacterium that lives in deep ocean waters near hot sea vents. This enzyme is used in PCR because it works best in extremely hot surroundings. The optimal temperature for functioning is 75-80°C. Also, it works fast and can replicate a 1000 base pair DNA strand in under 10 seconds at 72°C.


 * __Forward Primer__**- also known as the upstream primer, the forward primer is a nucleotide sequence. It binds to a single strand of DNA, and by doing this, allows for the DNA polymerase (or Taq polymerase) to bind and initiate replication. Nucleotides will be added in the 5' to 3' direction, and the replication will occur towards the opposing "reverse" primer. In the PCR reaction, these primers will be carefully chosen to anneal to the DNA strand on either side of the gene of interest. Therefore, when the replication occurs, the process will be made more efficient because, effectively, the only DNA being copied is the gene of interest. No nucleotides, and more importantly no time will be wasted.

__**Reverse Primer**__- also known as the downstream primer, the reverse primer is a nucleotide sequence. Similar to the Forward Primer, it anneals to the DNA and allows for replication by serving as a binding site for polymerases.

__**dNTPs (Nucleotides)**__- also known as deoxyribonucleoside triphosphates, but simply put "nucleotides" are key in the process of PCR. Nucleotides--adenine, thymine, guanine and cytosine--are the building blocks of DNA molecules. DNA molecules are built of millions of nucleotide base pairs that are connected with hydrogen bonds. The temperature increase in the PCR process denatures the double helix such that the two antiparallel DNA strands unravel and split. Then, after the primer pairs anneal to the strands, the Taq polymerase binds and replicates the DNA by attaching free nucleotides to the strands. A and T compliment, and G and C compliment. Thus, through this process, two DNA strands result from one parent strand in a "semi-conservative" manner.

__**Target DNA (Template)-**__ the target DNA strand was extracted from the worms in our specific lab. The DNA was taken, and because we knew the specific genetic sequence of our gene of interest (bli-1) we were able to find specific primers that surrounded our gene sequence. Only our specific area of our target DNA was replicated, which is the beauty of PCR. Only one double-stranded piece of target DNA is needed. Every cycle of PCR yields twice the number of strands of the previous cycle. Therefore, one strand of target DNA will shortly becomes a million copies within a few hours or less.


 * __Buffer-__** in a standard procedure of PCR, a buffer is needed and must contain Mg--the cofactor for the polymerase. However, in we used the Taq 2X master mix, which already contained dNTP's, Taq polymerase and the necessary buffer. Therefore, the simple Taq 2x Master Mix was used without the need for additional buffer.

=// The Rough Game Plan //=

The polymerase chain reaction consists of three main steps repeated over and over in succession that effectively clones a very specific gene of interest. A very important, and possibly hindering aspect of PCR, is that the genetic sequence of the target gene must be known beforehand. When this is known however, the sequence is harnessed to create over a billion copies from a single fragment of DNA--just imagine if your average copy machine could turn out a billion copies with such ease!

//**The process is simple and relies chiefly on the manipulation of temperature**// 1. The temperature is raised to roughly 90-95 degrees Celsius--the double stranded DNA will denature. The hydrogen bonds that held the two helices together will break, and the double stranded DNA will split into two single stranded DNA. Note that because of the incredible outnumbering of primers to DNA ratio, the DNA strand does not recoil on itself when the temperature is finally dropped.

2. The temperature is dropped to roughly 50-55 degrees Celsius--the primers, which have been specifically chosen to bind to the target gene, are able to anneal to the DNA when the temperature is dropped.

3. The temperature is raised to roughly 72 degrees--the Taq polymerase is now able to adhere to the primers and then elongate the DNA strand in the 5' to 3' direction. The Taq polymerase is specifically chosen because of its ability to function at high temperatures (see "Active Players" for details about Taq polymerase). When these three steps are repeat continually for many cycles, the DNA strands double every time, and thus the growth follows the exponential growth pattern of (#target gene fragments) = (#original target gene fragments) X 2^(# of cycles).

//**However, there are many details that must be considered**// 1.The process does involve knowledge of the target gene's genetic sequence (i.e. the arrangement of nucleotides). When choosing the pair of primers, each primer has its own specific temperature that it operates best at. Therefore, the one must consider trying to match the forward and reverse primer as close as possible together for optimal results. Otherwise, when the temperature is lowered in step two, only one primer might anneal and lead to errors in replication.

2. When the primer pair is being designed, if the gene is intended to be inserted into a vector (such as L4440 in our case) then the ends of the primers need to be able to be digested in order to create the desired "sticky ends." This process is simple. Every enzyme (in our case SacII and HindIII) has its specific sequence that it looks to cut at. With knowledge of the restriction enzymes, the same nucleotide sequence can be literally manufactured. Therefore, with simple knowledge of what the genetic sequence of the target gene of interest is, and with simple knowledge of what restriction enzymes will be used in the subsequent procedure, the primers with cut sites can be engineered at various standard primer design sites (see "Resources Used"). To see the primers that were designed specifically in our lab, and to read more Bli-1 related specifics, see "Our Process."

3. When designing primers, in order to raise the temperature that a primer operates at, the nucleotide ratio can be manipulated. (The amount of G and C nucleotides should be increased compared to the amount of A and T nucleotides.) At a certain temperature, the primers are unable to

=//// Results ////= Image 1: Explanation: This gel shows the results from the PCR that was run with the primers (see further below under "our specifics") designed specifically for Bli-1 gene. The right most lane is a 100 bp Ladder. This was chosen because our PCR product of interest is approximately 300 bp. Interpretation: All three PCR amplification tubes worked and produced product. The second tube produced the most PCR product. Image 2: Explanation: This gel shows much more than the PCR product. This gel was run to verify that the PCR and L4440 were digested successfully with the SacII and HindIII. The pAMP was indeed digested (though the label does not denote that) and was done so in order to verify the digestion took place. Note: when PCR was digested, because we designed the primers to have the cut sites at the end of the primers, the digestion of the PCR did not change the size by anything significant.



=// Our Specifics //= 1. Taq 2X Mastermix - the protocol below was found linked to the site where the Taq 2X Mastermix was purchased. (neb.com)

2. The specific genetic code for our gene of interest - bli-1 - was found on wormbase.org. The orange lettering symbolizes the nucleotide sequences that do not code for any specific gene at all, but merely fill space on the DNA molecule. These are called introns and are not transcribed. Regardless, our genetic sequence below was found and the primers were designed with online primer design sites. The following primer sequences were formulated by the online site when the above code was copy/pasted into their website program. Then, in order for the PCR product that results to be digested, the specific cut sites for SacII and HindIII were added to the ends of the primers. We designed specific primers in class with the oversight of Ms. Appel our teacher.

Forward Primer sequence : 5' - GGG GAA GCT TTT CAC ACC CTG GAT TTG GAC CTC A - 3' Reverse Primer sequence : 5' - AAA ACC GCG GAG GTG TTC CCG GAG AAC CAT TCA T - 3'

The temperatures for these primers were given on the label, and this temperature was programed into the PCR machine, a machine which systematically repeated the PCR process of changing temperatures at the exact time periods to yield PCR product.

Pictures:

=// Sources //= http://www.wormbase.org/ http://www.neb.com http://tools.invitrogen.com/ http://www.idtdna.com other primer design sites