In order to learn more about a particular gene, it is possible to artificially insert the DNA containing that gene into another DNA and allow the biology to take over and replicate the gene. Linear phage DNA can be cleaved into two parts. A foreign DNA insert can then be added between the two phage ends. When the phage DNA is used to carry another foreign DNA in this way, the phage DNA is said to be a vector.
In cloning you start out with phage vector DNA and cut (cleave) it in the middle. Human DNA (or any foreign DNA) containing the gene you want to study is cut into many pieces and mixed with the phage DNA. The phage DNA picks up pieces of human DNA and inserts them between two phage arms. Each little segment of human DNA is cloned into a separate phage DNA. DNA ligase allows the phage DNA and the foreign DNA to be joined together. When this new recombinant DNA is put back into the cell, the particular gene that you are interested in learning more about will be replicated as the DNA is replicated.
The collection of all the recombinant DNA molecules containing phage which contains segments from the entire (human) genome is said to be a recombinant DNA library. In order to create this library, the recombinant DNA is incorporated into virus particles and grown in a Petri dish containing a lawn of bacteria. Individual plaques will develop corresponding to each phage. The phage in each plaque are identical and carry a specific piece of human DNA. (See text Figure 4.10). In this manner the whole human genome can be cloned. Human DNA consists of about 6 billion base pairs, which in the process of cleavage are divided into about 300,000 fragments of 20,000 basepairs each. Each of those 300,000 fragments is then cloned into a vector.
Plasmid DNAs are a small circular DNAs in prokaryotic cells that can range in size from 2000-40,000 base pairs. Antibiotic resistance genes are often encoded on plasmid DNAs. Antibiotics are naturally occurring substances that kill bacteria. Any cell which contains a plasmid with an antibiotic resistance gene will provide the cell with the ability to grow in the presence of that particular antibiotic. This is why antibiotics must be taken for a certain amount of time even if symptoms have disappeared, because the bacteria that was still alive would be resistant to the antibiotic (All of the non-resistant bacteria would have been killed) and would replicate itself, creating more resistant bacteria.
Plasmid DNA can also be used as a vector. For example ampicillin resistance plasmids allows the cell to grow in the presence of ampicillin. Foreign DNA can be inserted in the plasmid DNA in the same way that foreign DNA could be inserted into the phage DNA as was discussed above. see text Figure 4.11.
Restriction enzymes recognize specific sequences on DNA, then cut the DNA at those sequences. There are about 2500 known restriction enzymes which have different specificities for cutting the DNA. A complete list is available in the REBase web site. see text Figure 4.12. Most of the sequences that are recognized by restriction enzymes are palindromic, meaning they read the same backward and forward. For example, the restriction enzyme BamH1 recognizes the DNA sequence GGATCC. The complementary strand of DNA, reading in the opposite direction, would also read GGATCC. This palindromic quality makes it easier for the restriction enzyme to recognize the sequence, since it is present on both strands of DNA.
Sometimes the restriction enzyme cuts straight through the DNA, cutting both strands at the same location. Most of the time, however, restriction enzymes cleave the DNA in a staggered cut - leaving a few nucleotides of single stranded DNA extending from the cut site. For example, the BamH1 restriction enzyme cuts both strands of DNA between the adjacent G's, leaving "sticky ends" of DNA on each strand:
5'-----G GATCC-------------CCTAG G-----5'
These sticky ends allow separate DNA molecules to get together. The short sticky ends actually can base pair between two different DNAs to align the two DNA molecules. Any two DNAs cut with the same restriction enzyme will have the same sticky ends and therefore can be joined. It is this ability provided by restriction enzymes that allows most of recombinant DNA techniques to work.
BamH1 will recognize this sequence and cut DNA approximately once in every 4096 basepairs (46). If a plasmid is cut by a restriction enzyme, it will also have sticky ends and could base pair with pieces of human DNA. In this way it is possible to create a plasmid library similar to the way it is done with phage vectors.
Once the DNA has been cut into pieces by restriction enzymes, different sized DNA molecules can be separated by means of gel electrophoresis. The DNA is pushed by an electric field through a gel (a three dimensional matrix of fibers that are all tangled together - the texture of a gel is just like Jell-O, another gel). The big DNA molecules move slowly because they have trouble getting through the net. The small molecules, however move right through. Intermediate sized molecules move at an intermediate rate. Thus the different sizes are separated and can appear as discrete bands on the gel. This is useful to isolate a particular piece of DNA.
To create a human library using plasmid DNA vectors involves transforming cells with DNA and then selecting those cells in the population that have picked up the recombinant DNAs. To generate the recombinant DNA library, you mix together linearized plasmid DNAs and human DNAs and incubate the mixture under conditions that will facilitate forming recombinant DNAs. After incubating, the mixture will contain some recombinant DNA molecules, some non-recombined plasmid DNA and some plain old human DNA. This mixture is then incubated with E. coli under conditions that favor the bacteria picking up DNA from their environment.
From this mixture of bacteria containing different DNAs, you want to isolate only those that have picked up recombinant DNA molecules. The trick one can use is to utilize a plasmid that contains two antibiotic resistance genes: an ampicillin resistance gene, and a tetracycline resistance gene - the insertion point for the foreign DNA is in the middle of the tetracycline gene. The four possible different kinds of bacteria in this mixture can then be represented as having the following antibiotic resistance traits:
The idea now is to select only those bacteria containing recombinant DNA. Ampicillin will kill any non-resistant bacteria. Tetracycline will stop non-resistant bacteria from growing but will not kill them. To perform the selection do the following:
If you want to find a particular gene within your library you go through a process called screening the library. For example, let's detail the process used to find the globin gene. First, the phage library is spread out on a series of petri dishes containing bacterial lawns. Plaques are allowed to form, each plaque representing a specific fragment of cloned DNA. A nitrocellulose disc is then overlaid on each petri dish and then carefully peeled off from the surface of the dish. This yields a replica of the phage plaques in the exact same location as the original petri dish.
The disc is treated to remove proteins, to denature the DNA and then to anchor the DNA to the filter. Next a radioactively-labeled nucleic acid from red blood cells (a radioactive probe) is hybridized to each filter disc. The probe (globin mRNA) will hybridize only to its complementary DNA sequence on the filter. The unbound probe is removed and the disc is exposed to x-ray film.
Only the DNA from the plaque that has hybridized to the probe will expose the x-ray film. This allows the particular clone containing the desired gene to be identified and then grown up by picking phage from the original petri dish from which the filter was made. see text Figure 5.3.
If you are just interested in the mRNA sequences, it is possible to clone them alone. An enzyme called reverse transcriptase is used to make a complementary DNA by synthesizing DNA starting from the poly(A) tail on the mRNA. This complementary DNA (cDNA) runs the along the mRNA and creates a double stranded molecule that has one DNA strand (the cDNA) and one RNA strand (the mRNA) paired to each other. Through a number of enzymatic and chemical steps the RNA is replaced by DNA in this hybrid molecule, resulting in a double stranded cDNA molecule (a "complementary DNA")
The advantage of using this approach is that one can create a cDNA library of only those sequences that are expressed in a given tissue. Only those genes which are expressed will produce mRNA. It is possible to create tissue specific cDNA libraries: a liver cDNA library, kidney cDNA library, tumor cDNA library. By studying the composition of the cDNA library it is possible to gain some insight into gene expression in different tissues.
Another useful feature of cDNA clones is that they do not contain any introns, only exons, since they were made from an mRNA template. All introns are removed during RNA processing. By comparing the sequence of a cDNA clone with the sequence of the corresponding genomic clone, it is possible to determine the location and size of all of the introns in a gene. Finally cDNAs can be used in expression vectors.