Page 32 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

326 Chapter 9 Digital Analysis of DNA

cell may end up with hundreds of identical copies of the recombinant DNA molecules into host cells also with 100%
same plasmid molecule. efficiency, the resulting set of clones would represent the
Each viable plasmid-containing bacterial cell will mul- entire genome in a fragmented form. A hypothetical collec-
tiply to produce a distinct spot on an agar plate, consisting tion of cellular clones that includes one copy—and one
of a colony of tens of millions of genetically identical cells. copy only—of every sequence in the entire genome would
The colony as a whole is considered a cellular clone. Such be a single complete genomic library.
clones can be identified when they have grown to about How many clones are present in this hypothetical li-
1 mm in diameter. The millions of identical plasmid mole- brary? If you started with the 3,000,000 kb of DNA from
cules contained within a colony together make up a DNA a haploid human sperm and reliably cut it into a series of
clone (Fig. 9.5b). 150 kb restriction fragments, you would generate
3,000,000/150 = 20,000 genomic fragments. If you
placed each and every one of these fragments into
Libraries Are Collections BAC cloning vectors that were then transformed into
of Cloned Fragments E. coli host cells, you would create a perfect library of
20,000 clones that collectively carry every locus in the
Moving step by step from the DNA of any organism to a genome. The number of clones in this perfect library
single purified DNA fragment is a long and tedious pro- defines a genomic equivalent. To find the number of
cess. Fortunately, scientists do not have to return to step 1 clones that constitute one genomic equivalent for any
every time they need to purify a new genomic fragment library, you simply divide the length of the genome (here,
from the same organism. Instead, they can build a genomic 3,000,000 kb) by the average size of the inserts carried by
library: a long-lived collection of cellular clones that con- the library’s vector (in this case, 150 kb).
tains copies of every sequence in the whole genome in- In real life, it is impossible to obtain a perfect li-
serted into a suitable vector (Fig. 9.6). Like traditional brary. Each step of cloning is far from 100% efficient,
book libraries, genomic libraries store large amounts of and the DNA of a single cell does not supply sufficient
information for retrieval upon request. They make it possi- raw material for the process. Researchers must thus har-
ble to start a new cloning project at an advanced stage, vest DNA from among the millions of cells in a particular
when the initial step of recombinant DNA construction has tissue or organism. If you make a genomic library with
already been completed and the only difficult task left is to this DNA by collecting only one genomic equivalent
determine which of the many clones in the library contains (20,000 clones for a human library in BAC vectors),
the DNA sequence of interest. Once the correct cellular then by chance some human DNA fragments will appear
clone is identified, it can be amplified to yield a large more than once, while others will not be present at all.
amount of the desired genomic fragment. Including four to five genomic equivalents produces an
If you digested the genome of a single cell with a re- average of four to five clones for each region (locus) of
striction enzyme and ligated every fragment to a vector the genome, and a 95% probability that any individual
with 100% efficiency, and you then transformed all of these locus is present at least once.

Figure 9.6 Part of a human genomic DNA library. Each essential concepts
colony on these plates contains a different recombinant plasmid
with a different fragment of the human genome. • To form recombinant DNA molecules, DNA ligase links
© McGraw-Hill Education. Lisa Burgess, photographer together restriction-enzyme-cut vector and genomic DNA
fragments with the same sticky ends.
• Vectors include an origin for DNA replication and a
selectable marker such as a gene for antibiotic
resistance. Plasmid vectors are used for small DNA
inserts (<20 kb), while BAC or YAC vectors can carry
much larger inserts.
• Recombinant DNAs are transformed into host cells.
When a single transformed cell is grown into a cellular
clone, each cell in the clone has the same recombinant
DNA.
• Genomic libraries contain a species’ entire genome as a
collection of random DNA fragments; multiple genome
equivalents are needed to ensure representation of all
parts of the genome.