2009 Mayıs | Turkish Chemistry
May 17

Protocols

 

This site gives you general information on how and why molecular techniques work. Here are links to some sites that give you specific protocols for performing individual techniques.

May 17

Sequencing

Sequencing

One of the major methods of sequencing in known as chain termination sequencing, dideoxy sequencing, or Sanger sequencing after its inventor biochemist Frederick Sanger.

The method is elegantly simple.
While chains are normally made up of deoxynucleotides (dNTPs), the Sanger method uses dideoxynucleotides.

dideoxynucleotide and deoxynucleotide


Dideoxynucleotides (ddNTPs) are missing a hydroxy (OH) group at the 3′ position. This position is normally where one attaches to another to form a chain. If there is no OH group in the 3′ position, the additional nucleotides cannot be added to the chain, thus interrupting chain .

Manual sequencing

    1. Begin the process by synthesizing a chain that is complimentary to the template you want to analyze.

    2. Add a specific primer that you know will anneal.


    d

    3. Divide your sample among four test tubes. One test tube will be used for each specific (dGTP, dATP, dCTP, and dTTP).

    4. Add polymerase to each tube and one specific per tube.

    5. Add ddNTPs to all four tubes. Once you add the ddNTPs, there is no way for the chain to keep elongating, hence you have dd chain termination.

    6. Run this on a polyacrylamide gel using one lane per (dGTP, dATP, dCTP, and dTTP).

    7. To sequence, read the order of bases from the smallest to the largest.

Automated sequencing

Or, if this looks like too much work, you can pay for automated sequencing, where a machine does most of the work for you. Of course, you’ll need about $100,000 for the machine.

An automated sequencer runs on the same principle as the Sanger method (dideoxynucleotide chain termination). A laser constantly scans the bottom of the gel, detecting bands as they move down the gel. Where the manual method uses radioactive labeling, automated sequencing uses fluorescent tags on the ddNTPs (a different dye for each ). This makes it possible for all four reactions (dGTP, dATP, dCTP, and dTTP) to be run in one lane, so you can have huge numbers of reactions on one gel. This is a very efficient method.

It is important to remember, however, that a computer can make mistakes. Don’t trust the computer. Always check your printout for accuracy. You are looking for a good signal, at least in the 100s, and proper spacing, ideally about 12. Look also for big gaps between the bases since the computer can miss bases. It may often miss a G after an A, especially after an AA.

May 17

Plasmids

A plasmid is an independent, circular, self-replicating DNA that carries only a few genes. The number of plasmids in a cell generally remains constant from generation to generation. Plasmids are autonomous molecules and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using bacteria (see also alkaline lysis)

They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid is the basis of recombinant DNA technology. Plasmids

A plasmid is an independent, circular, self-replicating DNA that carries only a few genes. The number of plasmids in a cell generally remains constant from generation to generation. Plasmids are autonomous molecules and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.

Plasmids are easy to manipulate and isolate using bacteria (see also alkaline lysis) They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid is the basis of recombinant DNA technology.

There are two categories of plasmids. Stringent plasmids replicate only when the chromosome replicates. This is good if you are working with a protein that is lethal to the cell. Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to chromosome.

So how do we manipulate these plasmids?

    1. Mutate them using , ligation enzymes, and PCR. Mutagenesis is easily accomplished by using to cut out portions of one genome and insert them into a plasmid. PCR can also be used to facilitate mutagenesis. Plasmids are mapped out indicating the locations of their origins of replication and restriction enzyme sites.

    2. Select them using genetic markers. Some bacteria are antibiotic resistant. While this is a serious health problem, it is a godsend to molecular scientists. The gene that confers antibiotic can be added (ligated) to the gene you are inserting into the plasmid. So every plasmid that contains your target gene will not be killed by antibiotics. After you transfect your bacterial cells with your engineered plasmid (the one with the target gene and the antibiotic resistant marker), you incubate them in a nutrient broth that also contains antibiotic (usually ampecillin). Any cells that were not transfected (this means they do not have your target gene in them) are killed by the antibiotic. The ones that do have the gene also have the antibiotic resistant gene, and therefore survive the selection process.

    3. Isolate them (such as with alkaline lysis)

    4. Transform them into cells where they become vectors to transport foreign genes into a recipient organism.

There are some minimum requirements for plasmids that are useful for recombination techniques:

    1. Origin of replication (ORI). They must be able to replicate themselves or they are of no practical use as a vector.

    2. Selectable marker. They must have a marker so you can select for cells that have your plasmids.

    3. Restriction enzyme sites in non-essential regions. You don’t want to be cutting your plasmid in necessary regions such as the ORI.

In addition to these necessary requirements, there are some factors that make plasmids either more useful or easier to work with.

    1. Small. If they are small, they are easier to isolate (you get more), handle (less shearing), and transform.

    2. Multiple restriction enzyme sites. More sites give you greater flexibility in cloning, perhaps even allowing for directional cloning.

    3. Multiple ORIs. It is important to note that two genes must have different ORIs if they are going to be inserted in the same plasmid.

 

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