Chp 8 DETECTION OF GENES AND GENE PRODUCTS Huseyin Tombuloglu PhD. GBE310, Spring 2015.
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Transcript of Chp 8 DETECTION OF GENES AND GENE PRODUCTS Huseyin Tombuloglu PhD. GBE310, Spring 2015.
Chp 8
DETECTION OF GENES AND GENE PRODUCTS
Huseyin Tombuloglu PhD. GBE310, Spring 2015
• Molecular geneticists usually want to study particular genes within the chromosomes of living species– This presents a problem, because chromosomal DNA
contains thousands of different genes– The term gene detection refers to methods that distinguish
one particular gene from a mixture of thousands of genes
• Scientists have also developed techniques to identify gene products– RNA that is transcribed from a particular gene– Protein that is encoded in an mRNA
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DETECTION OF GENES AND GENE PRODUCTS
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A DNA library is a collection of thousands of different cloned fragments of DNA made by cutting up the genome of an organism When the starting material is chromosomal DNA, the
library is called a genomic library
A cDNA library contains hybrid vectors with cDNA inserts Should represent the genes expressed in the cells the RNA was
isolated from
The construction of a DNA library is shown in Figure 18.7
DNA Libraries
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18-46Figure 18.7
18-47Figure 18.7
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In most cloning experiments, the ultimate goal is to clone a specific gene
For example, suppose that a geneticist wishes to clone the rat b-globin gene Only a small percentage of the hybrid vectors in a DNA
library would actually contain the gene Therefore, geneticists must have a way to distinguish
those rare colonies from all the others
This can be accomplished by using a DNA probe in a procedure called colony hybridization Refer to Figure 18.8
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18-49Figure 18.8
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But how does one obtain the probe? If the gene of interest has been already cloned, a piece of
it can be used as the probe If not, one strategy is to use a probe that likely has a
sequence similar to the gene of interest For example, use the rat b-globin gene to probe for the b-globin
gene from another rodent
What if a scientist is looking for a novel type of gene that no one else has ever cloned from any species?
If the protein of interest has been previously isolated, amino acid sequences are obtained from it
The researcher can use these amino sequences to design short DNA probes that can bind to the protein’s coding sequence
18-50
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Southern blotting can detect the presence of a particular gene sequence within a mixture of many It was developed by E. M. Southern in 1975
Southern blotting has several uses 1. It can determine copy number of a gene in a genome 2. It can detect small gene deletions that cannot be
detected by light microscopy 3. It can identify gene families 4. It can identify homologous genes among different
species
Southern Blotting
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Prior to a Southern blotting experiment, the gene of interest, or a fragment of a gene, has been cloned
This cloned DNA is labeled (e.g., radiolabeled) and used as a probe
The probe will be able to detect the gene of interest within a mixture of many DNA fragments
The technique of Southern Blotting is shown in Figure 18.9
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An alternative type of transfer uses a
vaccuum
Figure 18.9
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a) The steps in Southern blotting
A common labeling method is the use of the radioisotope 32P
Conditions of high temperature or high salt concentrations
Probe DNA and chromosomal fragment must be nearly
identical to hybridize
Temperature and/or ionic strength are lower
Probe DNA and chromosomal fragment must be similar but not necessarily identical to hybridize
Gene of interest is found only in single copy in the genome
Gene is member of a gene family composed of three
distinct members
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Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules It was not named after anyone called Northern! Originally known as ‘Reverse-Southern’ which became Northern.
Northern blotting has several uses 1. It can determine if a specific gene is transcribed in a
particular cell type Nerve vs. muscle cells
2. It can determine if a specific gene is transcribed at a particular stage of development
Fetal vs. adult cells 3. It can reveal if a pre-mRNA is alternatively spliced
Northern Blotting
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Northern blotting is rather similar to Southern blotting It is carried out in the following manner
RNA is extracted from the cell(s) and purified It is separated by gel electrophoresis It is then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a
radioactive probe The filters are then exposed to an X-ray film
RNAs that are complementary to the radiolabeled probe are detected as dark bands on the X-ray film
Figure 18.10 shows the results of a Northern blot for mRNA encoding a protein called tropomyosin
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Figure 18.10
Smooth and striated muscles produce a larger amount of tropomyosin mRNA than do brain cells
This is expected because tropomyosin plays a role in muscle contraction
The three mRNAs have different molecular weights This indicates that the pre-mRNA is alternatively spliced
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Western blotting is used to identify a specific protein within a mixture of many protein molecules Again, it was not named after anyone called Western!
Western blotting has several uses 1. It can determine if a specific protein is made in a
particular cell type Red blood cells vs. brain cells
2. It can determine if a specific protein is made at a particular stage of development
Fetal vs. adult cells
Western Blotting
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Western blotting is carried out as follows: Proteins are extracted from the cell(s) and purified They are then separated by SDS-PAGE
They are first dissolved in the detergent sodium dodecyl sulfate This denatures proteins and coats them with negative charges
The negatively charged proteins are then separated by polyacrylamide gel electrophoresis
They are then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a primary
antibody (recognizes the protein of interest) A secondary antibody, which recognizes the constant
region of the primary antibody, is then added The secondary antibody is also conjugated to alkaline phosphatase
The colorless dye XP is added Alkaline phosphatase converts the dye to a black compound
Thus proteins of interest are indicated by dark bands18-59
18-60
Figure 18.11 shows the results of a Western blot for the b-globin polypeptide
This experiment indicates that b-globin is made in red blood cells but not in brain or intestinal cells
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Researchers often want to study the binding of proteins to specific sites on a DNA molecule For example, the binding to DNA of transcription factors
To study protein-DNA interactions, the following two methods are used 1. Gel retardation assay
Also termed band shift assay 2. DNA footprinting
Techniques that Detect the Binding of Proteins to DNA
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Figure 18.12
The technical basis for a gel retardation assay is this: The binding of a protein to a fragment of DNA retards its rate of
movement through a gel
Gel retardation assays must be performed under nondenaturing conditions
Buffer and gel should not cause the unfolding of the proteins nor the separation of the double helix
Higher mass and therefore slow migration
Lower mass and therefore fast migration
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DNA footprinting was described originally by David Galas and Albert Schmitz in 1978 They identified a DNA site in the lac operon that is bound
by the lac repressor This DNA site is, of course, the operator
The technical basis for DNA footprinting is this: A segment of DNA that is bound by a protein will be
protected from digestion by the enzyme DNase I
Figure 18.13 shows a DNA footprinting experiment involving RNA polymerase holoenzyme
18-63
18-64Figure 18.13
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Did not ontain RNA pol holoenzyme
18-65Figure 18.13
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In the absence of RNA pol holoenzyme, a
continuous range of sizes occurs
No bands in this range
RNA pol holoenzyme is bound to this DNA region, and thus
protects it from DNase I
Thus RNA pol holoenzyme binds to an
80-nucleotide region
(from -50 to +30)
• Analyzing and altering DNA sequences is a powerful approach to understanding genetics
– A technique called DNA sequencing enables researchers to determine the base sequence of DNA
• It is one of the most important tools for exploring genetics at the molecular level
– Another technique known as site-directed mutagenesis allows scientists to change the sequence of DNA
• This too provides information regarding the function of genes
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18.3 ANALYSIS & ALTERATION OF DNA SEQUENCES
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During the 1970s two DNA sequencing methods were devised One method, developed by Alan Maxam and Walter
Gilbert, involves the base-specific cleavage of DNA The other method, developed by Frederick Sanger, is
known as dideoxy sequencing
The dideoxy method has become the more popular and will therefore be discussed here
DNA Sequencing
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The dideoxy method is based on our knowledge of DNA replication but uses a clever twist
DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one and the 3’–OH of the other (Refer to Fig. 11.10)
Nucleotides missing that 3’–OH can be synthesized
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Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow
This is referred to as chain termination If ddATP is used, termination will always be at an A in the DNA
Figure 18.14
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Prior to DNA sequencing, the DNA to be sequenced must be obtained in large amounts
This is accomplished using cloning or PCR techniques
In many sequencing experiments, the target DNA is cloned into the vector at a site adjacent to a primer annealing site
In the experiment shown in Figure 18.15, the vector DNA is from a virus called M13
After cloning, the viral DNA is introduced into the host cell There it will produce single-stranded DNA as part of its life cycle
If double-stranded DNA is used as the template, it must be denatured at the beginning of the experiment
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Figure 18.15
The newly-made DNA fragments can be separated according to their length by
running them on an acrylamide gel
They can then be visualized as bands when the gel is exposed to X-ray film
Sequencing ladder
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An important innovation in the method of dideoxy sequencing is automated sequencing
It uses a single tube containing all four dideoxyribonucleotides
However, each type (ddA, ddT, ddG, and ddC) has a different-colored fluorescent label attached
After incubation and polymerization, the sample is loaded into a single lane of a gel
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Figure 18.16
The procedure is automated using a laser and fluorescent detector
The fragments are separated by gel electrophoresis Indeed, the mixture of DNA fragments are electrophoresed off the end
of the gel As each band comes off the bottom of the gel, the fluorescent
dye is excited by the laser The fluorescence emission is recorded by the fluorescence detector
The detector reads the level of fluorescence at four wavelengths
18-72Figure 18.16
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Analysis of mutations can provide important information about normal genetic processes Therefore, researchers are constantly looking for mutant
organisms
Mutations can arise spontaneously, or be induced by mutagens
Researchers have recently developed techniques to make mutations within cloned DNA
Site-Directed Mutagenesis
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One widely-used method is known as site-directed mutagenesis It allows the alteration of a DNA sequence in a specific way
The site-directed mutant can then be introduced into a living organism
This will allow the researchers to see how the mutation affects The expression of a gene The function of a protein The phenotype of an organism
Mark Zoller and Michael Smith developed a protocol for the site-directed mutagenesis of DNA cloned in a viral vector Refer to Figure 18.17
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Figure 18.17
The vector is the M13 virus which produces a
single-stranded DNA as part of its life cycle
Depending on which base is replaced, the mutant or original sequence is produced
Can be identified by DNA sequencing and
used for further studies