Answer to Question #170490 in Molecular Biology for Johnae Renard

Question #170490

A sample from the planet Mars has yielded a new eukaryotic organism, the MARS Amoeba (MARSA). Initial studies have determined it contains a nucleus with 3 distinct DNA based chromosomes. You have been assigned the task to study this new creature.


Using your knowledge, you are expected to: (25 points each)


1)   Generate both MARSA genomic and cDNA libraries while testing your libraries for completeness and titer by using many techniques discussed in our lectures such as Southern and Northern Blotting, mRNA to cDNA techniques, genomic DNA isolation via Principle of pulse-field gel electrophoresis (PFGE) followed by partial restriction digestions, and ligations into lambda phage vectors (expression-based Lambda gt11.). Include in your written journey all need steps and controls.

Expert's answer

Genomic library

1. Extract and purify DNA.

The technique of pulsed gel electrophoresis is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA.

2. Digest the DNA with a restriction enzyme. This creates fragments that are similar in size, each containing one or more genes.

A given restriction enzyme cuts DNA segments within a specific nucleotide sequence, at what is called a restriction site. These recognition sequences are typically four, six, eight, ten, or twelve nucleotides long and generally palindromic (i.e. the same nucleotide sequence in the 5' – 3' direction). Because there are only so many ways to arrange the four nucleotides that compose DNA (Adenine, Thymine, Guanine and Cytosine) into a four- to twelve-nucleotide sequence, recognition sequences tend to occur by chance in any long sequence. Restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories, and as a result, several potential "restriction sites" appear in almost any gene or locus of interest on any chromosome.

After restriction digest, DNA can then be analysed using agarose gel electrophoresis (Southern Blotting). In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (literally pipetted into small wells at one end of the slab). The gel is then subjected to an electric field, which draws the negatively charged DNA across it. The molecules travel at different rates (and therefore end up at different distances) depending on their net charge (more highly charged particles travel further), and size (smaller particles travel further). Since none of the four nucleotide bases carry any charge, net charge becomes insignificant and size is the main factor affecting rate of diffusion through the gel.

3. Insert the fragments of DNA into lambda vector that was cut with the same restriction enzyme. Use the enzyme DNA ligase to seal the DNA fragments into the vector. This creates a large pool of recombinant molecules.

To prepare infectious λ virions carrying recombinant DNA, the phage-assembly process is carried out in vitro. In one method, E. coli cells are infected with a λ mutant defective in A protein, one of the two proteins required for packaging λ DNA into preassembled phage heads. These cells accumulate preassembled “empty” heads; since tails attach only to heads “filled” with DNA. Preassembled tails also accumulate in these cells. An extract containing high concentrations of empty heads and tails is prepared by lysing cells infected with the λ A mutant. When this extract is mixed with isolated A protein (obtained from λ-infected cells) and recombinant λ DNA containing a COS site, the DNA is packaged into the empty heads. The tails in the extract then combine with the filled heads, yielding complete virions carrying the recombinant λ DNA.

4. These recombinant molecules are taken up by a host bacterium by transformation, creating a DNA library.

Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.[43] Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are called competent cells.

The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.

DNA entry into E. coli cells is through channels known as zones of adhesion or Bayer's junction, with a typical cell carrying as many as 400 such zones. Their role was established when cobalamine (which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.[44]

It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.

Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms.

5. Testing for titer.

Dilutions of the library are used to transform cultures of E. coli of known concentrations. The cultures are then plated on agar plates and incubated overnight. The number of viral plaques are counted and can be used to calculate the total number of infectious viral particles in the library. Most viral vectors also carry a marker that allows clones containing an insert to be distinguished from those that do not have an insert. This allows researchers to also determine the percentage of infectious viral particles actually carrying a fragment of the library.

cDNA library

The differense between genomic and cDNA library construction is that in first step we need to extract mRNA instead of DNA and construct cDNA using mRNA template.

mRNA extraction

Firstly, the mRNA is obtained and purified from the rest of the RNAs. Several methods exist for purifying RNA such as trizol extraction and column purification. Column purification is done by using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will bind. The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer and some heat to separate the mRNA strands from oligo-dT.

cDNA construction

Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymidine nucleotides) is tagged as a complementary primer which binds to the poly-A tail providing a free 3'-OH end that can be extended by reverse transcriptase to create the complementary DNA strand. Now, the mRNA is removed by using a RNAse enzyme leaving a single stranded cDNA (sscDNA). This sscDNA is converted into a double stranded DNA with the help of DNA polymerase. However, for DNA polymerase to synthesize a complementary strand a free 3'-OH end is needed. This is provided by the sscDNA itself by generating a hairpin loop at the 3' end by coiling on itself. The polymerase extends the 3'-OH end and later the loop at 3' end is opened by the scissoring action of S1 nuclease.




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