Download Cloning DNA and the construction of clone libraries Concepts

Lecture 25; 2007
Biology 207; Section B2; Good
Cloning DNA and the construction of clone libraries
Readings:
Griffiths et al (2000)
8th Edition: pp. 346-351; 7th Edition: Ch. 12 pp. 370-377
Assigned Problems: None.
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.
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:
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
Basic Steps for cloning a DNA fragment into a plasmid cloning vector
Fig 11-5 (8th) 12-5 (7th)
1) The foreign DNA is fragmented - usually with restriction enzymes - restriction
fragments with "sticky ends"
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Lecture 25; 2007
Biology 207; Section B2; Good
Restriction enzyme - cleaves DS DNA at specific nucleotide sequences
e.g. BamHI
5'.......GGATCC.......3'
3'.......CCTAGG.......5'
5'.......G GATCC.......3'
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 foreign (or target DNA).
"Sticky ends" - short stretch of complementary base pairs that anneal together and aid
in the formation of recombinant molecules.
e.g. Bam HI digestion foreign DNA fragment
3) Foreign fragment and opened vector are put together in the same 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) Cells with a plasmid within them are selected (Antibiotic resistance) and propagated
to 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
-> a clone.
A library is a collection of clones – Fig 11-6(8th) 12-5(7th)
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 real life - in the test tube - there are 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 duplicates.
Problems
1.- Restriction site positions are not always advantageous.- some too
big, too small,
2.- Order of fragments is not always easy to determine.
Solution: Construction of a genomic DNA library of "random clones" –Fig 11-8(8th) 28(7th).
Basic steps in constructing a cloning library:
1) isolate genomic cellular DNA
- partial digest - use 4-bp recognition restriction
enzymes - size select fragments
2) isolate cosmid vector DNA
3) ligate together to make recombinant molecules - shotgun
cloning idea
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Lecture 25; 2007
Biology 207; Section B2; Good
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.:
ABCD
--||----|--|-|----|---- Restriction map of clone
|---> |---> |---> |---> Genes
2- Start at random -> Show overlapping clones
3-Result is a map of all sequences.
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.
How many clones are needed in a library?
Class example had many more clones than the minimum needed.
Formula in the text 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.
Various vectors have different size foreign DNA inserts Vectors
pp. 346-350 (8th) 372-375(7th)
1) Plasmids - small segments of DNA can be cloned 0-10 Kb - circular DNA, with insert,
transformed into cell
2) Lambda phage - larger segments - 15-20 Kb per clone - use lambda in vitro
packaging system to put recombinant DNA into phage protein head then infect bacterial
host - more efficient. It is a linear DNA molecule.
3) Cosmid - larger inserts at 30-45 Kb per clone. This vector uses the lambda system to
transfer recombinant DNA into host E.coli cell but once inside it replicates with a
standard plasmid origin (circular DNA molecule).
The P1 cloning vector is a variation on cosmids using a different phage packaging
system that permits 80-100 Kb per clone and also uses a plasmid-like origin of
replication (circular DNA molecule).
4) BACs - Bacterial Artificial Chromosomes - Bacterial vector using a plasmid origin
of replication to propagate foreign DNA. The foreign DNA able to be cloned is 100's of
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Lecture 25; 2007
Biology 207; Section B2; Good
Kb, very, very large cloned regions
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