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 in the middle. Human DNA (or any foreign DNA) is cut into many pieces and mixed with the phage DNA. The phage DNA picks up pieces of human DNA and inserts it between two phage arms. Each little segment of human DNA is cloned into a separate phage DNA. These recombinant DNA molecules are encapsulated into virus particles. The collection of all the recombinant DNA containing phage which contains segments from the entire (human) genome is said to be a recombinant DNA library. In this case, since we started with genomic DNA, the library is said to be a genomic library. If the genomic phage library spread 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.Plasmid DNAs are a small circular DNAs that can range in size from 2000-40000 base pairs long. 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.
Plasmid DNA can also be used as vectors. 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.
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 three different kinds of bacteria 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:
Restriction enzymes cut DNA at specific sequences. There are about 2500 different restriction enzymes which have different specificities for cutting the DNA. see text Figure 4.12. Sometimes the restriction enzyme cuts straight through the DNA, cutting both strands at the same location. Most of the time, however, restriction enzymes cut the DNA in a staggered fashion - leaving a few nucleotides of single stranded DNA extending from the cut site. These sticky ends are key to allowing separate DNA molecules to get together. The short sticky ends actually can base pair between two DNA ends 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.
Different sized DNA molecules can be separated by means of gel electrophoresis. The DNA is pushed by and 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.
If you want to find a particular gene within your library you go through a process called screening the library. 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 radioactive probe is hybridized to each filter disc. The probe will hybridize only to it's complementary DNA sequence. The unbound probe is removed and the disc is exposed to x-ray film. The DNA from the plaque that has annealed 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.
Messenger RNAs can also be used to create recombinant clones.
To start, the mRNA population is hybridized with an oligo(dT) - this is a short (15-20) DNA made up entirely of Ts (called deoxy-thymidines, or dT). The oligo(dT) will basepair with the poly(A) tail found on
mRNAs. Next an enzyme called reverse transcriptase is used to make a complementary DNA by synthesizing DNA starting
from the oligo(dT) that is basepaired to the poly(A) 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.
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 a 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. 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.
Expression vectors allow you to express certain genes directly from their recombinant DNAs. A typical expression vector will have a promoter upstream of the DNA containing the sequence to be expressed. Usually the "gene" is a cDNA because if the gene contained introns the introns would not be removed in bacteria. In addition, the promoter that is used can be an inducible one, so that synthesis of the gene product can be regulated. For example, if you wanted to express human growth hormone, you might place the growth hormone cDNA downstream of a lac promoter. In the presence of lactose, bacteria containing this construct will produce human growth hormone.
Shuttle vectors allow DNAs to be transferred between two different species. The shuttle vector has two origins of replication: one that works in each host. Typically, one host is bacterial and is used for all of the cloning steps and the other host is a eukaryotic organism that can be used to study the expression from that cloned gene or can be used to synthesize a product from the gene.
Shuttle vectors can be used to perform what has been called reverse genetics. It is possible to replace or alter the sequence of regulatory elements that control expression of a given gene then put gene back into their normal host cells to see how gene behavior has changed. This provides information on how the regulatory element might function and which are the important sequences within the regulatory element itself.
Another possibility is to put a regulatory element to be studied in front of a gene whose activity can be easily evaluated, such as the beta-galactosidase gene from the lac operon. There is a simple way to measure the level of beta-galactosidase in any cell - the enzyme turns a colorless substrate into a blue substance. Therefore, by adding this substrate to tissues, one can just measure the level of "blueness" to know the level of expression of the beta-galactosidase gene. The beta-galactosidase gene is being used as a reporter gene in this case because it "reports" on the activity of the promoter by which it is controlled. Reporter genes provide a powerful way of determining in which tissues and under which circumstances specific promoters are active.
One of the first things scientists do after isolating a specific fragment of DNA is to create a restriction map. A restriction map is simply a "map" of the locations of different restriction enzyme cut sites along the length of the DNA. Restriction maps are useful in further cloning of DNA fragments and for identifying specific regions in the cloned DNA (e.g. exons, introns, and promoters). Restriction maps can be created in sufficient detail that a restriction map can uniquely identify a piece of DNA as being different from any other piece of DNA.
Once a restriction map is produced, and it sufficiently shows the details of DNA, the next step is to discover where on the cloned DNA, the gene lies. It is also necessary to figure out if what is found is the entire gene sequence or only a fragment of it. We can do this through DNA blotting followed by hybridization with a probe. In this protocol the cloned DNA is digested with a specific restriction enzyme or with a set of restriction enzymes and then the different restriction fragments are separated by size using gel electrophoresis. After being separated, the fragments are transferred directly from the gel onto a nitrocellulose filter, producing a replica on the filter of the pattern of bands on the gel. In a process similar to that for screening a library, the filter is treated to denature the DNA and then to anchor the DNA to the filter. This filter is then hybridized with a radioactive probe containing the sequence of interest. For example, an experiment might use a radioactive pure mRNA to hybridize to a gene for that mRNA. In this case, only those restriction fragments containing exon sequence will hybridize. The hybridized regions on the nitrocellulose filter can be detected by exposing x-ray film.
Genomic blotting is a technique in which the starting material for the electrophoresis gel is whole genomic DNA rather than just the DNA from a cloned fragment. In this case, the probe will hybridize only to the restriction fragment(s) from the total genomic digest that contains its complementary sequence. Different individuals might have slight differences in their genes and this difference will be detected in genomic blotting of this type. For example, two individuals will both have insulin genes, but the two genes might have slightly different sequences either in the gene itself or in the flanking regions to the gene. This could lead to different restriction fragment sizes for different alleles of the same gene.
This difference can be used in forensics to identify individuals and can be used by scientists to identify similar genes from different species.