A useful class of enzymes are site-specific endonucleases, also known as restriction enzymes, that recognize and cut DNA molecules at or near short nucleotide sequences (typically four to eight base pairs in length). Figure FI (similar to Table 25.2 on page 933) lists some restriction endonucleases. The recognition sites typically show two-fold rotational symmetry, sometimes called a palindrome. Specific methylation (shown as asterisks in Figure F1) protects DNA from cleavage by the restriction endonuclease. Figure F2 shows that blunt cuts (= blunt or flush ends) or staggered cuts (= cohesive ends) can be created by endonuclease cleavage. Cleavage of phosphodiester bonds by restriction endonucleases creates 5'-phosphates and 3'-hydroxyls.
Note: Restriction endonuclease sites are really genetic markers; that is, a point mutation changing any given base pair in the site prevents cleavage of the site. For example, GAATTC is cut by EcoRI, but GTATTC is not (bold shows the nucleotide change; only the sequence of one strand is shown).
Restriction endonucleases cut DNA molecules into smaller fragments. The number of fragments and the sizes (typically expressed in base pairs, bp, or kilobase pairs, kb) of the fragments can be determined by gel electrophoresis (Figure F3). The order of the fragments gives a physical map of the DNA molecule (also known as a restriction map). Figure F4 (see also Figure 25.6 on page 931) shows one way to determine a physical map. The textbook shows another method for mapping restriction endonuclease sites on page 969 (see Figure 25A.1). The problem below uses yet another method to order the sites on DNA. See if you can figure out how to do the problem!
Do you know how to map restriction endonuclease sites on DNA? Test yourself!
Enzymes that join DNA molecules together are called DNA ligases. DNA ligases reform phosphodiester linkages between adjacent 5'-phosphates and 3'-hydroxyls using an energy cofactor. All cells have DNA ligases, but two ligases are particularly important: E. coli DNA ligase and bacteriophage T4 DNA ligase (Figure 24.24). T4 DNA ligase is the enzyme of choice in the laboratory for joining DNA molecules together. We will discuss the importance of DNA ligases later when we study DNA replication.
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| E. coli DNA ligase |
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Joins only cohesive ends | ||
| T4 DNA ligase |
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Joins cohesive ends and blunt ends | ||
Phosphates can be removed from the ends of nucleic acids by phosphatases (Figure F5). Phosphates can be added back to the ends of dephosphorylated nucleic acid by T4 polynucleotide kinase. T4 polynucleotide kinase transfers the gamma phosphate of ATP to the 5'-hydroxyl of nucleic acid. If the gamma phosphate is radioactive (32P), then the nucleic acid is labeled with the radioactive phosphate.
If a DNA fragment is joined to a self-replicating
DNA molecule, called a vector or vehicle, the recombinant DNA
molecule can be amplified, purified, and perpetuated forever.
There are many kinds of vectors: plasmids, viruses, transposons,
and even artificial chromosomes. Most vectors have been extensively
engineered to perform specialized functions. A common self-replicating
DNA molecule used in biotechnology is a plasmid (Figure
F6). Plasmids are extrachromosomal
(apart from the chromosome) pieces of circular DNA. Plasmids are
found in bacteria, yeast, and many other organisms as well. Plasmids
have a replication origin, and usually carry a few genes, especially
selectable genes ("markers") such as a gene conferring
resistance to an antibiotic. E. coli bacteria containing a plasmid
with a marker for resistance to ampicillin can be distinguished
from other E. coli not containing the plasmid with the marker
by growing the E. coli on a medium containing ampicillin. Such
a process is referred to as "selection" because one
is selecting the desired bacteria by using their desired trait
to identify them. Plasmids are used to carry foreign DNA into
bacteria (a process called transformation) for the purpose
of synthesizing the foreign protein, or for the study of the sequence
of the foreign DNA. The process of isolating a particular DNA
fragment by linking it to a vector is called DNA cloning (Note:
the process of isolating a gene is called "cloning"
a gene).
Linking DNA fragments to a vector
It is not quite so simple to join two DNA molecules together with ligase. Such a reaction is bimolecular and is therefore concentration dependent. Unfortunately, recircularization of individual DNA molecules is a unimolecular reaction which is not concentration dependent. There are several ways to prevent recircularization of linearized vector molecules. If the vector DNA is cut with two restriction enzymes with dissimilar cohesive ends, the DNA will not recircularize. Moreover, fragments of DNA produced by the same two enzymes can be inserted into the vector in a unique orientation. A more general approach is shown in Figure F7. The phosphates are removed from the ends of the linearized vector to prevent recircularization by ligase. DNA moolecules to be inserted into the vector retain phosphates at the ends. Thus, DNA fragments can be ligated to the vector. Any nicks will be repaired once the recombinant DNA molecules are put into cells (a process called transformation).
There are many ways to isolate a particular DNA fragment by DNA cloning. Researchers use the easiest cloning process because the isolation can take a few days to a few months depending on circumstances. We will examine two approaches: screening a library and genetic selection.
1. Screening a library
Let's assume you are interested in a growth hormone for chickens. You have purified the protein, but you want to obtain the gene. The protein can be sequenced to determine the order of the amino acids along the protein. Knowing the genetic code, the protein sequence can be "reverse translated" into DNA sequence (Figure F8). Since the Genetic Code is degenerate (more than one codon may specify a particular amino acid), there is some uncertainty in the predicted DNA sequence that codes for a protein; but if you are clever (and lucky), you can pick regions where the uncertainty is minimal. The sequences can be used to make an oligonucleotide probe that is labeled with radioactive phosphorous (Note - chemically synthesized oligonucleotides usually do not have a 5'-phosphate. Radioactive phosphate can be added to the 5'-end of the oligonucleotide using an enzyme called polynucleotide kinase and ATP with radioactive phosphate in the gamma position). Even radioactively labeled "degenerate" oligonucleotides can be used as probes to identify a particular DNA fragment in a mixture of fragments by stringent DNA hybridization. A collection of DNA fragments in a cloning vector is called a "library." For example, a genomic library is a collection of all the DNA sequences of an organism. A simple way to make a library is to cut genomic DNA into pieces with a restriction enzyme; in our example the genomic DNA is chicken DNA. The cut genomic DNA is ligated to vector DNA cut with the same enzyme: all of the genomic DNA fragments will be joined to vector DNA (ideally one genomic DNA fragment per vector DNA molecule). If one transforms this DNA mix into E. coli and then grows the cells on a plate containing the antibiotic to which the vector is resistant, one will obtain thousands-to-millions of colonies of bacteria, each containing the vector with one piece of the original chicken genome. This entire collection of bacterial colonies is called a library.
(Note: The bacteria forming a colony
are referred to as a "clone.")
The problem now is to identify the particular E. coli colony that
contains the particular piece of cloned DNA you want; in our example,
all or part of the chicken hormone gene. We can do this using
a method called colony hybridization (Figure F9). A master plate containing the library (collection
of colonies) is replica plated to a nitrocellulose membrane disk
placed on a nutrient plate. The colonies grow on the disk. The
nitrocellulose disk is removed from the plate and processed as
follows: (a) add NaOH to break open the bacteria and denature
the DNA released from the cells, (b) neutralize the NaOH, (c)
add protease to remove proteins, (d) wash the membrane, and (e)
bake the membrane at 80 C to immobilize the DNA onto the membrane.
The DNA on the membrane is then hybridized with a radioactive
probe under conditions where only perfectly base-paired double
stranded DNA sequences will be stable. During this process, the
probe will "find" the DNA complementary to it and form
a duplex. Other DNAs that are not complementary will not form
a duplex with the probe. The membrane is washed to remove all
of the non-hybridized probe. Finally, the membrane is placed on
a piece of X-ray film. After developing, the film contains one
or more spots corresponding to the regions of the film where the
desired bacterial clones are located. By examining the original
master plate, the correct clones containing all or part of the
chicken growth hormone gene can be identified.
2. Genetic selection
Sometimes the "properties" of a DNA fragment itself can be used to identify the fragment for easy cloning. The cloning of a DNA fragment containing the E. coli origin of DNA replication (the origin is a small DNA sequence in the E. coli chromosome that controls the start of DNA replication; it is called oriC) is an example of this strategy (Figure F10). Each "colony" of bacteria that were selected in this example arose from a single precursor parent bacterium that received a recombinant plasmid that had the ampicillin resistance gene as well as the E. coli origin of replication. The resulting plasmids are called oriC (for origin of the E. coli Chromosome) plasmids. These plasmids constitute mini E. coli chromosomes that are much easier to study. The minimum region acting as an origin is 245 bp long, is located at position 84 on the E. coli map, and is highly conserved in DNA sequence among other enterobacteria. A replicon is a self-perpetuating DNA molecule having an origin controlling DNA replication (cis-acting control sequence) and all of the genes necessary to replicate the DNA (trans-acting enzymes and "factors"). The E. coli chromosome is a replicon, and an oriC plasmid is a replicon as long as it is inside an E. coli cell (the cell chromosome, not the plasmid, supplies the trans-acting genes).
There are a few limitations to general cloning strategies. We will learn that eukaryotic genes may not only be very large (even millions of base pairs long), but come in pieces called exons and introns. Since only the exons code for proteins, "reverse translated" amino acid sequences that are adjacent to each other in the protein may actually span two or more exons that are distantly separated from each other in the genome. There are size limits for DNA cloning. Plasmid vectors can accomodate only DNA fragments under about 10,000 base pairs. Even though other specialized cloning vectors can accept DNA fragments up to and even exceeding 100,000 base pairs, dealing with very large, intact genes is still a formidable problem. Very large genes can be cloned in pieces using the method called chromosome walking (Figure F11). This can be thought of as the reiterative isolation of genomic fragments from a genomic library. By isolating overlapping clones, stretches of DNA that are one "step" away from each other can be identified and mapped. The initial probe may be designed from "reverse translated," minimally degenerate sequences as discussed above. The clones isolated using this initial probe are mapped with restriction enzymes. A new probe is selected from an end of one of the clones, and the library is screened again. This procedure is repeated many times until a collection of clones spanning the entire gene is assembled. This method can also be used to "walk" from a known gene to a neighboring unknown gene.