Page 44 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

338 Chapter 9 Digital Analysis of DNA

16. Suppose you are using a plasmid cloning vector that fraction of the genome in a single recombinant clone.
has no EcoRI sites (5′ G^AATTC 3′) in its polylinker (Note: ln is the natural log, sometimes written as log e .)
because the particular drug resistance gene your b. Calculate f for the genomic library described in part (a).
vector contains has an EcoRI site within it. c. How many different recombinant BAC clones
a. How could you use the following two oligonucle- would you need to have a 99% chance that a spe-
otides (and ligase enzyme) to ligate an insert cific 100,000 bp region of the genome is repre-
that is an EcoRI fragment into the BamHI site sented? How many clones for a 99.9% chance?
(5′ G^GATCC 3′) in the polylinker of your vector?
d. How many genomic equivalents correspond to each
of your answers in part (c)?
5' G A TC CG GGGG G GGG G 3'
5' A A TT CC CCCC C CCC G 3' e. Suppose that after you ligated the human DNA
inserts with the BAC vectors and transformed
b. How many EcoRI sites will the recombinant DNA E. coli with the mixture, you find that you have
contain? How many BamHI sites? only 30,000 drug-resistant colonies transformed
c. In part (a), you used the two oligonucleotides to with recombinant plasmids. What is the chance
make a so-called adapter. Adapters can also be that any specific 100,000 bp region of the genome
used to ligate blunt-ended inserts into vectors cut is represented in a recombinant plasmid?
with sticky-ended enzymes. Design an adapter that f. If you want to construct a complete human ge-
would allow you to ligate blunt-ended inserts into nomic library that contains the smallest number of
the BamHI site of your vector’s polylinker. independent recombinant clones possible, what is
(Note: Two blunt-ended DNA fragments can be li- the key variable that you should adjust?
gated together, although the reaction is much less
efficient than sticky-end ligation.) One difficulty in molecular cloning using plasmid vectors is
17. As a molecular biologist and horticulturist specializ- that the restriction enzyme-digested vector can be resealed
ing in snapdragons, you have decided that you need to by DNA ligase without an insert of genomic DNA. The next
make a genomic library to characterize the flower two problems investigate methods to deal with this issue.
color genes of snapdragons.
a. How many genomic equivalents would you like to 19. The lacZ gene from E. coli encodes the enzyme
β-galactosidase, which can catalyze the conversion of a
have represented in your library to be 95% confi- colorless compound called X-gal into a blue product.
dent of having a clone containing each gene in Molecular biologists have taken advantage of this prop-
your library? erty by constructing plasmid vectors that contain the
b. How do you determine the number of independent lacZ gene with an EcoRI site in its middle (see figure
clones that should be screened to guarantee this that follows). After cutting this vector with the EcoRI
number of genomic equivalents? enzyme, scientists ligate it together with EcoRI-
18. Suppose you are constructing a human genomic li- digested human genomic DNA, transform the resultant
brary in BAC vectors where the human DNA frag- molecules into ampicillin-sensitive E. coli cells, and
ments are on average 100,000 bp. plate these cells on petri plates containing ampicillin
a. What is the minimum number of different recom- and X-gal. Some of the colonies growing on this plate
binant BACs you need to construct in order to have are white in color, while others are blue. Why?
a greater than zero chance of having a complete
library—meaning one in which the entire genome
is represented? lacZ gene
The simple statistical equation that follows allows you EcoRI amp r
to determine the size that a genomic library needs to site
be (that is, the number of independent recombinant
clones you need to make) for a given likelihood that Origin of
replication
the entire genome is represented in the library.
ln (1 − P) 20. Your undergraduate research advisor has assigned
N = you a task: Insert an EcoRI-digested fragment of
ln (1 − f )
frog DNA into the vector shown in Problem 19.
In the equation, N is the number of independent recom- Your advisor suggests that after you digest your
binant clones; P is the probability that any particular part plasmid with EcoRI, you should treat the plasmid
of the genome is represented at least one time; f is the with the enzyme alkaline phosphatase. This enzyme