Page 29 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

9.2 Cloning DNA Fragments 323

the Human Genome Project. Sophisticated methods have sticky ends will form base pairs and the ligase will stabilize
been developed recently to circumvent the need for molec- the molecule by forming phosphodiester bonds between ad-
ular cloning in determining the sequence of genomes, and jacent nucleotides (one from the vector and one from the
some of these techniques will be described in later chap- genomic DNA insert).
ters. However, molecular cloning remains today an essen- Laboratory tricks can increase the efficiency and gen-
tial component of many important approaches to the eral utility of molecular cloning. For example, certain pro-
analysis and manipulation of DNA. cedures prevent two or more genomic fragments from
Molecular cloning consists of two basic steps. In the first, joining with each other rather than with the vectors. Other
DNA fragments are inserted into specialized chromosome- methods minimize the chance that vector molecules can
like carriers called cloning vectors, which ensure the reseal themselves without including an insert of genomic
transport, replication, and purification of individual DNA DNA. Yet other manipulations can be performed to con-
inserts. In the second step, the combined vector-insert mol- nect fragments of genomic DNA that do not have sticky
ecules are transported into living cells, and the cells make ends to vectors. These techniques ensure that researchers
many copies of these molecules. Because all the copies of can reliably produce the molecular clones they intend.
a given fragment are identical, the group of replicated DNA
molecules is known as a DNA clone. DNA clones may be
purified for immediate study or stored within cells or vi- Choice of vectors
ruses as collections of clones known as libraries for future Available vectors differ from one another in biological prop-
analysis. We now describe each step of molecular cloning. erties, carrying capacity, and the type of host they can infect.
Different types of vectors have different experimental uses.
The simplest vectors are small circles of double-stranded
Ligating Inserts to Vectors Produces DNA known as plasmids that can gain admission to and
replicate within many kinds of bacterial cells, independently
Recombinant DNA Molecules of the bacterial chromosomes (Fig. 9.4a). The most useful
On their own, small fragments of human genomic DNA can- plasmids contain a polylinker, which is a short, synthetic
not reproduce themselves in a cell. To make replication pos- DNA sequence that contains a number of different restric-
sible, it is necessary to splice each fragment to a vector. tion enzyme restriction sites (Fig. 9.4a). Each of these sites
Vectors must contain two kinds of specialized DNA se- is found once in the polylinker but nowhere else in the plas-
quences: one to provide a means of replication for the vector mid vector. The polylinker provides flexibility in the choice
and the foreign DNA inserted into it, and the second to sig- of enzymes that can be used to digest the DNA containing
nal the vector’s presence to an investigator by conferring a the fragment of interest. Exposure to any one of these restric-
detectable property on the host cell. A vector must also have tion enzymes opens up the vector at the corresponding rec-
distinguishing physical traits, such as size or shape, by which ognition site, allowing the insertion of a foreign DNA
it can be purified away from the host cell’s genome. Several fragment cut with the same enzyme, without at the same
types of vectors are in use, and each one behaves as a mini- time splitting the plasmid into many pieces (Fig. 9.4b and c).
chromosome capable of accepting foreign DNA inserts and Plasmid vectors can carry only relatively small foreign DNA
replicating independently of the host cell’s genome. The cut- fragments less than about 20 kb long.
ting and ligating together of vector and inserted fragment— Each plasmid vector carries an origin of replication
DNA from two different origins—creates a recombinant and a gene for resistance to a specific antibiotic (Fig. 9.4a).
DNA molecule. The origin of replication enables the plasmid to replicate
independently inside a bacterium. The gene for antibiotic
resistance confers on the host cell the ability to survive in a
Sticky ends and base pairing medium containing a specific antibiotic; the resistance
Two characteristics of sticky ends provide a basis for the gene thereby enables experimenters to select for propaga-
efficient production of a vector-insert recombinant: First, tion only those bacterial cells that contain a plasmid. Anti-
the single-strand overhangs are available for base pairing. biotic resistance genes and other vector genes that make it
Second, no matter what the origin of the DNA (bacterial or possible to pick out cells harboring a particular DNA mol-
human, for example), two sticky ends produced with the ecule are called selectable markers. Plasmids fulfill the
same enzyme are always compatible, that is, complemen- final requirement for vectors—ease of purification—
tary in sequence. because they can be purified away from the genomic DNA
To make recombinant DNA molecules, you simply cut of the bacterial host by several techniques that take advantage
the vector with the same restriction enzyme used to generate of size and other differences.
the fragment of genomic DNA, and then you mix the di- The largest-capacity vectors are artificial chromo-
gested vector and genomic DNAs together in the presence of somes: recombinant DNA molecules that combine replica-
the enzyme DNA ligase (Fig. 9.4). The complementary tion and segregation elements in such a way that they