Classical gene cloning and identification

The basic approach to cloning a segment of DNA entails:

1. Initial enzyme-based fragmentation of intact genomic DNA (usually chromosomes isolated as described earlier in this chapter) so that it is broken down into manageable fragment sizes for further manipulation. Ideally all/most fragments will contain one gene.

2. Integration of the various fragments generated into cloning vectors, which are themselves small DNA molecules capable of self-replication. Typically, these are plasmids or viral DNAs and the composite or engineered DNA molecules generated are called rDNA.

3. Introduction of the vectors housing the DNA fragments into host cells.

4. Growing these cells on agar plates.

5. Screening/identification of the host cell colonies containing the rDNA molecules (i.e. screening the 'library' of clones generated) in order to identify the specific colony containing the target DNA fragment, i.e. the target gene (Figure 3.12).

We will now look at each of these stages separately. The initial fragmentation of genomic DNA is undertaken using enzymes known as restriction endonucleases (REs). Some 800 different REs have been identified thus far. These enzymes recognize, bind and cut DNA sequences which exhibit a defined base sequence (Table 3.2). These sequences normally exhibit a twofold symmetry around a specific point and are usually 4, 6 or 8 bp in length. Such areas are often termed palindromes. In general, the larger the recognition sequence the fewer such sequences present in a given DNA molecule and, hence, the smaller the number of DNA fragments that will be generated. Depending upon the specific RE utilized, DNA cleavage may yield blunt ends (e.g. BsaAI and EcoRV in Table 3.2) or staggered ends - the latter are often referred to as sticky ends.

An essential feature of the cloning vector used is that it must be capable of self-replication in the cell into which it is introduced, which is usually E. coli. Two of the most commonly used types of vector in conjunction with E. coli are plasmids and bacteriophage X. Plasmids are circular extra-chromosomal DNA molecules, generally between 5000 and 350 0000 bp in length, that are found naturally in a wide range of bacteria. They generally house several

Vectors

rDNA molecules

Host cell i

Colony 3 contains the cloned gene of interest

Colony 3 contains the cloned gene of interest

Agar plate containing 3 colonies,each is a clone of one of the host cells above

Figure 3.12 A basic overview of the DNA cloning process. Refer to text for specific details

genes, often including one or more genes whose product renders the plasmid-containing cell resistant to specific antibiotic(s). One plasmid often used in cloning experiments with E. coli is pUC18 (Figure 3.13). Bacteriophage ('phage') are viruses capable of infecting and replicating inside bacteria. Bacteriophage X DNA is approximately 48 500 bp in length. Another vector type sometimes used are the bacterial artificial chromosomes (BACs), which are effectively very large plasmids used to clone very large stretches of DNA (usually DNA fragments above 100 000 bp).

Integration of the DNA fragments into the chosen vector is undertaken by 'opening up' the circular vector via treatment with the same RE as used to generate the DNA fragments for cloning, followed by co-incubation of the cleaved vector and the fragments under conditions that promote the annealing of complementary sticky ends. Some vectors may simply recircularize to reform their original structure, but pretreatment of the vector in various ways can prevent this from happening. Most of the recircularized plasmids will have incorporated a fragment of DNA to be cloned. The plasmids are then incubated with another enzyme, a DNA ligase, which catalyses the formation of phosphodiester bonds in the DNA backbone and thus will seal or 'ligate' the plasmid.

The next stage of the cloning process entails the introduction of the engineered vector into E. coli cells. This can be achieved by a number of different means. One approach (called transformation) involves co-incubation of the plasmids and cells in a solution of calcium chloride, initially at 0 °C, with subsequent increase in temperature to 42 °C. This temperature shock facilitates entry of plasmids into some cells.

Table 3.2 Some commercially available REs, their sources, DNA recognition sites and cleavage points

Restriction enzyme Source

DNA recognition sequence and cleavage sitea

BclI

BglII

BsaAI

BsaJI

BsiEI

EcoRV

MwoI

Tsp509I

XbaI

XhoI

Bacillus caldolyticus

Recombinant E. coli carrying BglII gene from Bacillus globigii Recombinant E. coli carrying BsaAI gene from Bacillus stearothermophilus A B. stearothermophilus J

B. stearothermophilus

Recombinant E. coli carrying EcoRV

gene from the plasmid J62 pig 74 Recombinant E. coli carrying cloned MwoI

gene from Methanobacterium wolfeii Thermus sp.

Recombinant E. coli carrying XbaI gene from Xanthomonas badvii Recombinant E. coli carrying XhoI gene from X. holcicola

5'-GCNNNNNlNNGC-3'

5'4AATT-3'

aG: guanine; C: cytosine; A: adenine; T: thymine; Pu: any purine; Py: any pyrimidine; N: either a purine or pyrimidine. Arrow indicates site of cleavage.

aG: guanine; C: cytosine; A: adenine; T: thymine; Pu: any purine; Py: any pyrimidine; N: either a purine or pyrimidine. Arrow indicates site of cleavage.

Figure 3.13 The plasmid pUC18 is often used for cloning purposes. It contains three genes: the ampicillin resistance gene (ampR), the lacZ gene, which codes for the enzyme p-galactosidase, and the lacI gene, which codes for a factor that controls the transcription of lacZ. Also present is an origin of replication (ori), essential for plasmid replication within the cell. Note the presence of a short stretch of DNA called the polylinker region located within the lacZ gene. The polylinker (also called a multiple cloning site) contains cleavage sites for 13 different REs. This allows genetic engineers great flexibility to insert a DNA fragment for cloning into this area. The polylinker has been designed and positioned within the lacZ gene so as not to prevent the expression of functional p-galactosidase. However, if a piece of DNA for cloning is introduced into the polylinker region, then the increased length does block p-galactosidase expression. The full sequence of the 2.69 kb plasmid is known and sequence analysis confirms the presence of multiple additional RE sites outside the polylinker region. There are at least six target sites for commonly used restriction enzymes within the ampR gene

Figure 3.13 The plasmid pUC18 is often used for cloning purposes. It contains three genes: the ampicillin resistance gene (ampR), the lacZ gene, which codes for the enzyme p-galactosidase, and the lacI gene, which codes for a factor that controls the transcription of lacZ. Also present is an origin of replication (ori), essential for plasmid replication within the cell. Note the presence of a short stretch of DNA called the polylinker region located within the lacZ gene. The polylinker (also called a multiple cloning site) contains cleavage sites for 13 different REs. This allows genetic engineers great flexibility to insert a DNA fragment for cloning into this area. The polylinker has been designed and positioned within the lacZ gene so as not to prevent the expression of functional p-galactosidase. However, if a piece of DNA for cloning is introduced into the polylinker region, then the increased length does block p-galactosidase expression. The full sequence of the 2.69 kb plasmid is known and sequence analysis confirms the presence of multiple additional RE sites outside the polylinker region. There are at least six target sites for commonly used restriction enzymes within the ampR gene

E colihousing plasmid in which DNA

E. c0lih0st cell E coliihoSt housing fragment has inserted into the lacZgene

Cells will not grow on agar plates containing ampicillin

Cells will grow on agar plates containing Ampicillin and X-gal and colonies will be blue in color

Cells will grow on agar plates containing ampicillin and X-gal and colonies will be a normal white color

Figure 3.14 Identification of E. coli host cell clones containing rDNA using pUC18 vectors. After transformation the cells are spread on agar plates containing ampicillin and a chemical called X-gal. Any untransformed cells present will fail to grow on these plates as the host E. coli cells contain no ampicillin resistance gene. This step, therefore, identifies cells into which plasmid has successfully been transferred (cell types (b) and (c)). Cells containing plasmid into which no foreign DNA has been inserted (cell type (b)) will grow on ampicillin-containing plates as the plasmid contains the ampR gene. This cell type also produces functional P-galactosidase (Figure 3.13). This enzyme will cleave X-gal, liberating a product that is blue in colour. Colonies, therefore, appear blue. Cells containing a plasmid in which a DNA fragment has been inserted into the lacZ gene (cell type (c), i.e. the desired cells) do not produce P-galactosidase; therefore, colonies derived from these cells will be a normal white colour

The E. coli cells are next spread out on the surface of an agar plate and incubated under appropriate conditions in order to kill cells that have not taken up plasmid. Each individual cell will thus form a colony (clone of cells). Three main types of cell will be initially transferred onto these agar plates: (a) some cells will have failed to take up any plasmid; (b) some transformed cells may have a plasmid in which no foreign DNA had been inserted; (c) some cells will house a plasmid that does carry a fragment of the target DNA. These latter cells are the only ones of interest, and various strategies may be adopted to identify them. One such strategy is outlined in Figure 3.14. Once the various E. coli clones (colonies) containing vector into which DNA fragments have been successfully integrated (i.e. clones containing rDNA) have been identified, all that remains to be achieved is to pinpoint which colony harbours the rDNA fragment containing the gene of interest (see Figure 3.12).

Assuming you started off with whole genomic DNA, the procedure thus far has effectively generated a library of clones containing different genomic DNA fragments. The final task remaining, therefore, is to identify which specific clone/clones harbour the actual DNA fragment of interest (in our context this would be the fragment containing the gene coding for the desired therapeutic protein). This can be a major task, as libraries often consist of 109 or more clones. The most common means of achieving this is via sequence-based hybridization studies. The basic approach taken entails the use of a labelled (e.g. radioactive) probe that is a single-strand DNA fragment or an RNA fragment synthesized to have a base sequence complementary to a sequence within the gene of interest. Genome projects now mean that such sequence information is known for many proteins. Alternatively, likely base sequences can be deduced if a partial amino acid sequence of the protein is known. Hybridization studies are usually initiated by physically pressing a nitrocellulose paper onto the agar plates containing the recombinant colonies. A replica of the plate is thus created on the paper, as some cells from each colony adhere to it. Subsequent treatment of the paper with alkali lyses the cells, releasing and denaturing the DNA within. The DNA adsorbs tightly to the paper. The paper is then exposed to a solution containing the labelled DNA probe under conditions that allow it to anneal to the target DNA, if it is present. After washing (to remove unbound probe), any probe retained on the paper surface can de detected by an appropriate visualization technique (e.g. autoradiography if the probe is radiolabelled), and the positioning of the label on the paper surface pinpoints which colony on the agar surface houses the desired DNA fragment. Cells from the appropriate colony can then be grown up in larger amounts by submerged fermentation (see Chapter 5) in order to produce larger amounts of the desired (now cloned) gene. The cells can be collected, lysed and the vector therein recovered by standard microbiological techniques. The cloned gene can then be excised from the vector via treatment with an appropriate RE and purified by standard molecular techniques.

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