2/15/16
BIOLOGY 207 - Dr. Locke
Lecture#22 - Cloning DNA and the construction of clone
libraries
Required readings and problems:
Reading: Open Genetics, Chapter 8
Problems: Chapter 8
Optional
Griffiths (2008) 9th Ed. Readings: pp 716-724
Problems: 9th Ed. None
Campbell (2008) 8th Ed. Readings: Concept 20.1, 20.2
Concepts:
How are DNA clone libraries made?
1. DNA segments from an organism can be cloned into vectors that replicate both the
organisms DNA segment and the vector DNA in a bacterial host cell.
2. Each independent assembly of a DNA segment in a vector is called a clone.
3. A collection of many different DNA segments, or clones, makes up a clone library
and can contain every DNA segment (or sequence) from the organism.
4. Gene libraries can be constructed in a variety of vectors.
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
page 1
2/15/16
Basics of cloning a segment of DNA
Recombinant DNA - a composite DNA molecule created in vitro by joining a foreign
DNA with a vector DNA molecule.
Recombinant DNA techniques - techniques for joining DNA molecules in vitro and
introducing them into cells (usually bacteria) where the molecules are
replicated.
"Recombining" DNA into cloning vectors - Recombine DNA fragments
-> is a form of recombination - generates recombinant DNA molecule.
Plasmids
- small circular DNA molecules
(about 2-10Kb) that contain:
Features:
1) Origin of replication -similar in function to
oriC in E. coli chromosome
2) Selectable marker gene - usually an
antibiotic resistance gene - e.g. ampR, tetR, kanR
3) Multiple cloning site (MCS) – many restriction enzyme sites in a short sequence.
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
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2/15/16
Cloning a DNA fragment into a plasmid cloning vector
Fig 20-2, 20.4 Griffiths (9th)
Basic Steps:
1) The target DNA is fragmented - usually with restriction enzymes
-> restriction fragments with "sticky ends"
Restriction enzyme - cleaves Double Stranded (DS) DNA at specific nucleotide
sequences e.g. BamHI
"Sticky ends" - short stretch of complementary base pairs that anneal together and
aid in the formation of recombinant molecules.
5'.......GGATCC.......3'
5'.......G
GATCC.......3'
3'.......CCTAGG.......5'
3'.......CCTAG
G.......5'
2) The circular plasmid vector DNA is opened, usually with a restriction enzyme that
has the same "stick ends" as the target DNA.
e.g. Bam HI digestion
target DNA fragment
.......G GATC C.......
.......CCTAG G.......
Biol207 Dr. Locke’s section
....G GATC C…target...G GATC C......
....C CTAG G...Insert....C CTAG G......
Lecture#22
Fall'11
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2/15/16
3) Foreign fragment and opened vector are put together in the same test tube and
ligated together - chemical process - random chance.
DNA Ligase seals the broken strands (total of 4) to produce recombinant
molecules
4) Recombinant molecules are transformed into a bacteria host cell
(remember Griffiths - transforming principle).
5) Transformed cells, with a plasmid within them, are selected (Antibiotic resistance)
and propagated -> amplify the number of plasmid molecules.
6) Amplified recombinant Plasmid DNA molecules can be purified and used for further
analysis
Note: each bacterial cell, and its descendants, will be derived from one original
recombinant DNA molecule
-> genetically identical
-> a clone.
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
page 4
2/15/16
A library is a collection of clones - Fig
In real life:
There are many fragments of foreign DNA.
Each can be cloned into a vector molecule. -> recombinant molecules
 - each is transformed into a different host cell
 - get a set of clones -> a library.
In the test tube -> many (106 -109) molecules -> many molecules joining together
which transform many cells.
At the end -> many clones 10's, 100's, 1000's etc.
**** This library would have many duplicate cloned fragments.
Problems with basic method:
1.- Restriction site positions are not always advantageous.
- some fragments too big, some too small, to clone.
2.- Order of fragments can not always be determined.
Need a method to clone random fragments --->
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
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2/15/16
Solution:
Construction of a genomic DNA library of "random clones"
- Fig
Basic steps in constructing a cloning library:
1) isolate genomic cellular DNA
- partial digest - use 4-bp restriction enzymes -> size select fragments
2) isolate cosmid vector DNA
3) ligate together to make recombinant molecules - shotgun cloning idea
4) plate out transformed E. coli - make a library of cosmid clones.
Come back to "random clones" idea -> partial digest will produce fragments that
overlap
Class Exercise on Random Genomic DNA Library
1- handout papers with clone regions on them -> each student is a clone.
Each 4 letter clone represents a fragment of a larger genome (alphabet).
The 4 letters denote a particular, and unique, position of the genome. e.g.:
A
B
C
D
--||----|--|-|----|---- Restriction map of clone
|---> |---> |---> |---> Genes
2- Start at random -> Show overlapping clones permit assembly of contig
3-Result is a map of all sequences.
Keep paper with letters!
Aberrant clones = chimeric clones that contain two or more regions of a genome,
which are not adjacent to each other in the genome, in one clone.
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
page 6
2/15/16
How many clones are needed in a library?
Class example had many more clones than the minimum needed.
Formula: P=1(1-f)n
Essentially says for a 99% chance at finding a specific unique sequence (e.g. any
letter in the "alphabet") one needs to look at 5X the number of clones that
minimally spans the genome.
From this: clone number is important and clone size is also important.
Example:
cosmid library of the E. coli genome
E. coli genome = about 4,500,000 bp
Cosmid clone contains 45,000 bp
4.5 x 106 / 4.5 x 104 = 1 X 102 = 100
Therefore one needs 100 clones for 1 genome equivalent
(minimal set of clones that could span the genome).
To get a 99% statistical probability of finding it one needs ~5 genome equivalents
(if it is a random chance).
Because clones are made at random, to get 99% chance of finding a specific
sequence or gene one needs 5 x 100 or 500 cosmid clones.
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
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2/15/16
Various vectors have different size foreign DNA inserts Vectors
1) Plasmids - small circular DNA vectors
- can clone 0-10 Kb fragments
- easily transformed into cell
2) Lambda phage - linear DNA vector
- can clone 15-20 Kb fragments
- uses lambda in vitro packaging system to put recombinant DNA into phage
protein head then infect bacterial host -> more efficient.
3) Cosmid – larger circular DNA vectors
- can clone 30-45 Kb per clone.
- uses efficient Lambda packaging system to transfer recombinant DNA into host
E. coli cell but replicates with a standard plasmid origin (circular DNA molecule).
4) BACs - Bacterial Artificial Chromosomes – circular DNA vector
- uses a plasmid origin of replication to propagate
- insert DNA able to be cloned is 100's of Kb, very, very large cloned regions
- very difficult to transform
Biol207 Dr. Locke’s section
Lecture#22
Fall'11
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