2/17/16
BIOLOGY 207 - Dr. Locke
Lecture#24 - Isolation of Genes from Libraries Using DNA Probes
Required readings and problems:
Reading: Open Genetics, Chapter 8
Problems: Chapter 8
Optional
Griffiths (2008) 9th Ed. Readings: pp 724-730
Problems: 9th Ed. Ch. 20: 17
Campbell (2008) 8th Ed. Readings: Concept 20.1, 20.2
Concepts:
How can DNA probes be used to find genes of interest?
1. The process of hybridization involves single DNA strands forming sequence specific
duplex of DNA.
2. Clones containing a particular gene, or DNA sequence, can be identified in a clone
library by using the process of hybridization and labeled DNA probes.
3. DNA probes from "natural" and "artificial" sources can be used but both rely on the
formation of DNA-DNA hybridization to make a sequence specific probe.
4. The basic procedure for clone identification is similar for screening most clone
libraries, and is also similar to probing Southern blots.
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Principle of DNA Hybridization
The use of DNA probes to recover clones from genomic, or other libraries, relies on
the principle of
DNA hybridization.
DNA is a duplex. Each strand is held to the other via hydrogen bonds between bases
in a base pair. Base stacking interactions help.
The strands can be separated, or pulled apart in a process called denaturation.
5'....GAATTCGGATCC....3' duplex DNA
3'....CTTAAGCCTAGG....5'
When denatured by heat (e.g. boiling water 100°C) or alkali (e.g. 50 mM Na0H) the
two single strands come apart (denature) to give:
5'....GAATTCGGATCC....3' and
3'....CTTAAGCCTAGG....5'
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Reassociation and annealing
In solution, these two single strands will come into contact (random process) and
anneal to reform the DNA duplex.
This occurs spontaneously in solution with the appropriate pH, salt concentration, and
temperature, etc.
The reassociation process depends upon the complementary sequences being able to
reform their original base pairs. So A must pair with T, G with C.
Sequences that are not complementary will not form base pairs and thus will not form
DNA duplexes.
Forming a Hybrid -> The process is sequence specific!
When the two strands reform a duplex, a hybrid is formed, hence the name
hybridization .
5'....GAATTCGGATCC....3' duplex DNA again
3'....CTTAAGCCTAGG....5'
Hybrids can form between
 DNA/DNA (two complementary DNA strands),
 DNA/RNA (a DNA strand and its complementary RNA sequence), or between
 RNA/RNA (not useful here since clones are DNA).
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Mis-match sequences can form hybrids, too
Hybrid formation only requires that the complementary sequences be similar (not
necessarily a perfect complementary sequence).
In a region where two strands begin to pair, and a hybrid can form, some "mis-match"
in the pairing sequences is permitted.
5'....GAATTTAGATCC....3' duplex DNA again
||||| |||||
3'....CTTAAGCCTAGG....5'
The extent of mis-match possible in a hybrid depends upon the hybridization
conditions (temperature, salt concentration etc).
Under some conditions 30-40% mis-matched strands are able to form a stable hybrid.
-> useful for cross species detection of genes.
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
DNA Probes
"Labeling" involves putting a tag on the DNA in some manner that permits one to
detect its presence, in minute quantities, at some later point.
DNA can be "labeled" in many different ways.
One widely used technique is to replace the normal Phosphorous of the DNA with a
radioactive atom of Phosphorous, 32P (normal = 31P)
This radioactivity can be detected by photographic emulsion, e.g. auto-radiography
onto sheets of film.
A cloned DNA sequence will hybridize to only its complementary sequences and thuse
provides an almost unique probe.
Labeling DNA
--P--P--P--P-label -32P--32P--32P--32P-G A T C
up
G
A
T
C
- one or both strands can be labeled.
- E.g. - Random priming method, nick-translation
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Sources of DNA for use as Probes
DNA for probes can come from a wide variety of sources, which can be divided into
two main categories:
Natural & Artificial
Natural
1. A similar gene from another organism
- Sequence of homologous genes is often similar enough to form a stable hybrid.
2. cDNA clone- Fig
A complementary (copy) DNA clone (usually a plasmid) that contains a cloned DNA
sequence derived from the reverse transcription of an mRNA sequence.
cDNA clones can be recovered from cDNA clone libraries by a variety of different
methods.
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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cDNA library: collection of cDNA clones, usually from one tissue/cell/organism
type.
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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Artificial
1. Synthetic oligo-nucleotide
- chemically synthesize a specific DNA sequence
(e.g the reverse translation of a protein sequence - the amino acid sequence can
be determined by biochemical techniques).
- the degeneracy of the triplet code usually makes it impossible to obtain a unique
sequence -> instead, usually use a degenerate sequence.
Example:
Protein sequence: Met - Lys - Asn – Glu
Lysine
codon AUG - AAA - AAU - GAA
Asparigine,
alternate
- AAG - AAC – GAG
Glutamic Acid
ATG
ATG
AAA
AAT
GAA
AAG
AAC
GAG
|
|
|
use both bases
Use a degenerate oligo-nucleotide.
2. PCR products
See Figure 8.6 in OG text
- polymerase chain reaction
- method for biochemically synthesizing Duplex DNA from a sequence of DNA.
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Now we have the probe labeled and ready to screen a genomic library for a gene of
interest.
Procedure to screen a library of genomic clones
Steps using plasmid clones:
1. Plate out library - each colony on the bacterial plate is a clone.
2. Lift clones (DNA) onto Nitrocellulose filter. Fix the clone DNA onto the filter and
denature the clone DNA, so as to make it able to form hybrids with probe.
3. Place filter in a hybridization bag with solution containing labeled, denatured probe
DNA. Incubate to permit the probe strands to form hybrids with the clone strands.
4. Wash away unhybridized probe.
5. Expose probed filter to X-ray film (autoradiography) to detect the presence of clones
with labeled probe.
6. From the X-ray film determine which clone hybridized to the probe and recover that
clone for further analysis.
Goal of clone library screening:
- to identify and recover a clone(s) that has the complementary sequence to the probe
(e.g a specific gene sequence)
- this clone can then be used for further work (e.g. DNA sequencing)
Classroom demo…
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
page 10
2/17/16
Southern Blot (Southern transfer)
A Southern Transfer is similar to probing a Library filter probing.
Genomic DNA, digested with a restriction enzyme, produces fragments of many sizes.
The size of fragments reveals some information about the sequence (genes).
We can use a Southern blot to identify the size of a fragment for which we have a
probe.
Steps in the probing process are similar:
1. Sample of DNA digested with a restriction enzyme is separated in an agarose gel by
the process of gel electrophoresis.
2. DNA in the gel is transferred to a membrane (Nitrocellulose filter), fixed and
denatured -> single stranded and able to hybridize to probe.
3. As before - Place filter in a hybridization bag with solution containing labeled,
denatured probe DNA. Incubate to permit the probe strands to form hybrids with
the fragment's complementary strands.
4. As before - Wash away unhybridized probe.
5. As before - Expose probed filter to X-ray film (autoradiography) to detect the
presence of labeled probe.
6. Autoradiogram shows the size of restriction fragments that hybridize to the probe
sequence (compared to known size markers).
Goal of Southern Blot:
- to identify the size of a restriction fragment that has the complementary sequence
to the probe (e.g a gene sequence)
Note: - RNA can be separated by gel electrophoresis and probed in a similar manner --> Northern blot -> Determine the size of mRNA for a gene’s transcript
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
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2/17/16
Summary
1- Complementary stands can form hybrids (hybridization).
2- Hybridization of labeled probes can be used to screen libraries of clones to find
complementary sequences.
3- Similar methods can be used to find the size of restriction fragments in genomic
DNA.
Biol207 Dr. Locke’s section
Lecture#24
Fall'11
page 13