Chapter 5Chapter 5

Molecular Tools Molecular Tools for Studying for Studying Genes and Gene Genes and Gene ActivityActivity

5.1 Molecular Separations

Gel Electrophoresis Two-dimensional Gel Electrophoresis Ion Exchange Chromatography Gel Filtration Chromatography

What is gel electrophoresis?What is gel electrophoresis?

Gel electrophoresis is a method that separates Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical basis of size, electric charge, and other physical properties. properties.

Many important biological molecules such as amino Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, posses ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as exist in solution as electrically charged species either as cations (+) or anions (-). Depending on the nature of the cations (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to net charge, the charged particles will migrate either to the cathode or to the anode. the cathode or to the anode.

Figure 5.1 DNA gel electrophoresis

(a) Scheme of the method; (b) A photograph

of a gel after electrophoresis showing the

DNA fragments as bright bands. DNA binds

to a dye that fluoresces orange under

ultraviolet light, but the bands appear pink in

this photograph.

Figure 5.2 Analysis of DNA fragment size by gel electrophoresis. (a) Photograph of a stained gel of commercially prepared fragments after electrophoresis.

The bands that would be orange in a color photo show up white in a black-and-white photo taken with an orange filter. The size of the fragments (in bp) are given at right.

Figure 5.2 Analysis of DNA fragment Figure 5.2 Analysis of DNA fragment

size by gel electrophoresis.(b)size by gel electrophoresis.(b)

Graph of the migration of the DNA Graph of the migration of the DNA

fragments versus their sizes in base fragments versus their sizes in base

pairs.pairs.

The vertical axis is logarithmic rather The vertical axis is logarithmic rather

than linear, because the electrophoretic than linear, because the electrophoretic

mobility (migration rate) of a DNA mobility (migration rate) of a DNA

fragments inversely proportional to the fragments inversely proportional to the

log of its size. However, notice the log of its size. However, notice the

departure from this proportionality at departure from this proportionality at

large fragment sizes, represented by the large fragment sizes, represented by the

difference between the solid line (actual difference between the solid line (actual

results) and the dashed line (theoretical results) and the dashed line (theoretical

behavior). This suggests the limitations behavior). This suggests the limitations

of conventional electrophoresis for of conventional electrophoresis for

measuring the sizes of very large measuring the sizes of very large

DNAs.DNAs.

Figure 5.3 Pulsed-field gel electrophoresis of yeast chromosomes.

Identical samples of yeast chromosomes were electrophoresed in 10 parallel lanes and stained with ethidium bromide (EB). The bands represent chromosomes having sizes ranging from 0.2 Mb (at bottom) to 2.2 Mb (at top). Original gel is ablout 13 cm wide by 12.5 cm long.

Figure 5.4 SDS-polyacylamide Figure 5.4 SDS-polyacylamide

gel electrophoresis.gel electrophoresis.

Polypeptides of the Polypeptides of the

molecular masses shown at molecular masses shown at

right were coupled to dyes and right were coupled to dyes and

subjected to SDS-PAGE. The subjected to SDS-PAGE. The

dyes allow us to see each dyes allow us to see each

polypeptide during and after polypeptide during and after

electrophoresis. electrophoresis.

SummarySummary

DNAs, RNAs, and proteins of DNAs, RNAs, and proteins of various masses can be separated by gel various masses can be separated by gel electrophoresis. The most common gel used electrophoresis. The most common gel used in nucleic acid electrophoresis is agarose, in nucleic acid electrophoresis is agarose, but polyacrylamide is usually used in but polyacrylamide is usually used in protein electrophoresis. SDS-PAGE is used protein electrophoresis. SDS-PAGE is used to separate polypeptides according to their to separate polypeptides according to their masses.masses.

What is 2D Gel Electrophoresis?What is 2D Gel Electrophoresis?

This is a method for the separation and This is a method for the separation and identification of proteins in a sample by identification of proteins in a sample by displacement in 2 dimensions oriented at displacement in 2 dimensions oriented at right angles to one another. This allows the right angles to one another. This allows the sample to separate over a larger area, sample to separate over a larger area, increasing the resolution of each increasing the resolution of each component. component.

What is it used for?What is it used for?

22D gel electrophoresis is generally used as a D gel electrophoresis is generally used as a component of proteomics and is the step used component of proteomics and is the step used for the isolation of proteins for further for the isolation of proteins for further characterization by mass spectroscopy characterization by mass spectroscopy

for the large scale identification of all proteins for the large scale identification of all proteins in a sample. in a sample.

differential expression. differential expression.

How is it Performed?How is it Performed?

IEFIEF is used in the 1st Dimension (Righetti, is used in the 1st Dimension (Righetti, P.G., 1983). This separates proteins by their P.G., 1983). This separates proteins by their charge (pI).charge (pI).

SDS-PAGESDS-PAGE in the 2nd Dimension. This in the 2nd Dimension. This separates proteins by their size (molecular separates proteins by their size (molecular weight, MW).weight, MW).

The procedure is known as ISO-DALT: iso The procedure is known as ISO-DALT: iso for isoelectric focusing and dalt for dalton for isoelectric focusing and dalt for dalton weightweight

SummarySummary

High-resolution separation of High-resolution separation of polypeptides can be achieved by two-polypeptides can be achieved by two-dimensional gel electrophoresis, which dimensional gel electrophoresis, which uses isoelectric focusing in the first uses isoelectric focusing in the first dimension and SDS-PAGE in the second.dimension and SDS-PAGE in the second.

Ion exchange chromatographyIon exchange chromatography

In ion exchange chromatography, charged In ion exchange chromatography, charged substances are separated via column materials that substances are separated via column materials that carry an opposite charge. The ionic groups of carry an opposite charge. The ionic groups of exchanger columns are covalently bound to the gel exchanger columns are covalently bound to the gel matrix and are compensated by small matrix and are compensated by small concentrations of counter ions, which are present concentrations of counter ions, which are present in the buffer. When a sample is added to the in the buffer. When a sample is added to the column, an exchange with the weakly bound column, an exchange with the weakly bound counter ions takes place. counter ions takes place.

ION EXCHANGE CHROMATOGRAPHYION EXCHANGE CHROMATOGRAPHY

There are two commonly used ion exchangers, There are two commonly used ion exchangers, binding either anions or cations, attached to a solid binding either anions or cations, attached to a solid support material, such as hydrogel or cellulose. One is support material, such as hydrogel or cellulose. One is CMC or CM-cellulose. This is a -ve molecule attached to CMC or CM-cellulose. This is a -ve molecule attached to a inert support of cellulose. This is effective at pH >4. a inert support of cellulose. This is effective at pH >4. This is therefore an cation exchanger. The other is an This is therefore an cation exchanger. The other is an anion exchanger, the DEAE (DiEthylAminoEthyl). It anion exchanger, the DEAE (DiEthylAminoEthyl). It also has a cellulose support, but is a +ve molecule. It is also has a cellulose support, but is a +ve molecule. It is effective at pH <9. effective at pH <9.     The binding of proteins to these resins depends on 3     The binding of proteins to these resins depends on 3 properties- properties- *Ionic character of protein. *Ionic character of protein. *pH of buffer holding protein. *pH of buffer holding protein. *Ionic strength of solution/total salt conc. *Ionic strength of solution/total salt conc.

Commonly used functional groupsCommonly used functional groups Two exchanger types are differentiated: Two exchanger types are differentiated: basic basic

(positively charged) and (positively charged) and acidicacidic (negatively charged). (negatively charged). They in turn can be divided into those with weakly They in turn can be divided into those with weakly

basic or acidic character or strongly basic or acidic basic or acidic character or strongly basic or acidic character. character.

With strongly basic or acidic materials all functional With strongly basic or acidic materials all functional groups are always present in ionized form vastly groups are always present in ionized form vastly independent from the pH value in the specified independent from the pH value in the specified operating range. operating range.

For example, the quaternary amino groups (R3N+–) For example, the quaternary amino groups (R3N+–) are positively charged, while the sulfonic acid groups are positively charged, while the sulfonic acid groups (–SO(–SO33–) are negatively loaded. The pK values of the –) are negatively loaded. The pK values of the quaternary amino groups are around 14, those of the quaternary amino groups are around 14, those of the sulfonate residues below 1. sulfonate residues below 1.

In addition, weakly basic types (pK values between 8 In addition, weakly basic types (pK values between 8 and 11) and weakly acidic types (between 4 and 6) and 11) and weakly acidic types (between 4 and 6) exist. The weakly basic types consist of secondary exist. The weakly basic types consist of secondary and tertiary amino functional groups; the weakly and tertiary amino functional groups; the weakly acidic types of carboxyl functional group. Thus, a acidic types of carboxyl functional group. Thus, a weakly basic exchanger should only be used at pH weakly basic exchanger should only be used at pH values below 8.5, weakly acidic exchangers only at values below 8.5, weakly acidic exchangers only at pH values above 6. pH values above 6.

Outside these ranges strongly basic, or strongly Outside these ranges strongly basic, or strongly acidic exchangers should be used. Many proteins acidic exchangers should be used. Many proteins can be separated as polyanions (pH > pl) or as can be separated as polyanions (pH > pl) or as polycations (pH < pl), as long as the pH stability of polycations (pH < pl), as long as the pH stability of the protein of interest allows this selection. The most the protein of interest allows this selection. The most common ion exchanger groups are summarized in common ion exchanger groups are summarized in the table below together with their abbreviations the table below together with their abbreviations and pK valuesand pK values

Figure 5.6 Ion-exchange chromatographyFigure 5.6 Ion-exchange chromatography

Summary Summary

Ion-exchange chromatography can be Ion-exchange chromatography can be used to separate substances, including used to separate substances, including proteins, according to their charges. proteins, according to their charges. Positively charged resins like DEAE-Positively charged resins like DEAE-Sephadex are used for anion-exchange Sephadex are used for anion-exchange chromatography, and negatively charged chromatography, and negatively charged resins like phosphocellulose are used for resins like phosphocellulose are used for cation-exchange chromatography. cation-exchange chromatography.

Gel Filtration Chromatography Gel Filtration Chromatography Gel filtration chromatography is Gel filtration chromatography is

used to separate large molecules used to separate large molecules on the basis of size. Two on the basis of size. Two columns are run simultaneously. columns are run simultaneously. The first column contains The first column contains Sephadex G-75, which separates Sephadex G-75, which separates blue dextran and hemoglobin. blue dextran and hemoglobin. The second column contain The second column contain Sephadex G-10, which separates Sephadex G-10, which separates hemoglobin and riboflavin. hemoglobin and riboflavin. Because there is a difference in Because there is a difference in the two packing materials, the the two packing materials, the hemoglobin molecule runs very hemoglobin molecule runs very differently in the two columns. differently in the two columns.

Gel-filtration chromatography separates proteins on the basis of size. The technique measures the relative rates of passage through a molecular sieve. This molecular sieve is in the form of a polysaccharide gel in the shape of spherical beads.

Figure 5.7 Gel filtration chromatography. Figure 5.7 Gel filtration chromatography. (a)(a) Principle of the method. A resin bead Principle of the method. A resin bead is schematically represented as a “whiffle ball” (yellow). Large molecules (blue) cannot is schematically represented as a “whiffle ball” (yellow). Large molecules (blue) cannot fit into the beads, so they are confined to the relatively small buffer volume outside the fit into the beads, so they are confined to the relatively small buffer volume outside the beads. Thus, they emerge quickly from the column. Small molecules (red), by contrast, beads. Thus, they emerge quickly from the column. Small molecules (red), by contrast, can fit into the beads and so have a large buffer volume available to them. Accordingly, can fit into the beads and so have a large buffer volume available to them. Accordingly, they take a longer time to emerge from the column. they take a longer time to emerge from the column. (b)(b) Experimental results. Add a Experimental results. Add a mixture of large and small molecules from panel (a) to the column, and elute them by mixture of large and small molecules from panel (a) to the column, and elute them by passing buffer through the column. Collect fractions and assay each for concentration passing buffer through the column. Collect fractions and assay each for concentration of the large (blue) and small (red) molecules. As expected, the large molecules emerge of the large (blue) and small (red) molecules. As expected, the large molecules emerge earlier than the small ones. earlier than the small ones.

The column is eluted with buffer and the protein

concentration in the elute is measured.

V0 = Void volume: volume of buffer outside the beads.

This is the volume needed to elute the largest, completely excluded proteins.Ve = Elution volume: volume needed to elute any given

protein.Vt = Total volume: volume of buffer in the column, both

inside and outside the beads.

SummarySummary

Gel filtration chromatography Gel filtration chromatography uses columns filled with porous resins uses columns filled with porous resins that let in smaller substances, but exclude that let in smaller substances, but exclude larger ones. Thus, the smaller substances larger ones. Thus, the smaller substances are slowed in their journey through the are slowed in their journey through the column, but larger substances travel column, but larger substances travel relatively rapidly through the column. relatively rapidly through the column.

5.2 Labeled Tracers5.2 Labeled Tracers

Radioactive tracers can detect <10Radioactive tracers can detect <10-12 -12 gram gram of RNAof RNA

Techniques to detect radioactive tracers :Techniques to detect radioactive tracers :

AutoradiographyAutoradiography

Phosphorimaging Phosphorimaging

Liquid scintillation countingLiquid scintillation counting

Non-radioactive TracersNon-radioactive Tracers

5.2.1 Autoradiography5.2.1 Autoradiography

Autoradiography is a means of detecting radioactive Autoradiography is a means of detecting radioactive compounds with a photographic emulsion. compounds with a photographic emulsion.

Intensifying screenIntensifying screen

excited by β-rays excited by β-rays

at low temperature, at low temperature,

33H , H , 1414C, C, 3535SS DensitometerDensitometer

measures the absorbance of lightmeasures the absorbance of light

Figure 5.8 Autoradiography.Figure 5.8 Autoradiography.

(a)(a) Gel electrophoresis.Gel electrophoresis. Electrophorese Electrophorese radioactive DNA fragments in three parallel radioactive DNA fragments in three parallel lanes on a gel, either agarose or lanes on a gel, either agarose or polyacrylamide, depending on the sizes of the polyacrylamide, depending on the sizes of the fragments. At this point the DNA bands are fragments. At this point the DNA bands are invisible, but their positions are indicated here invisible, but their positions are indicated here with dotted lines. with dotted lines.

(b) Autoradiography.(b) Autoradiography. We place a piece of x- We place a piece of x-ray film in contact with the gel and leave it for ray film in contact with the gel and leave it for several hours, or even days if the DNA several hours, or even days if the DNA fragments are only weakly radioactive. Finally, fragments are only weakly radioactive. Finally, we develop the film to see where the we develop the film to see where the radioactivity has exposed the film. This shows radioactivity has exposed the film. This shows where the DNA bands are on the gel. In this where the DNA bands are on the gel. In this case, the large, slowly migrating bands are the case, the large, slowly migrating bands are the most radioactive, so the bands on the most radioactive, so the bands on the autoradiograph that correspond to them are the autoradiograph that correspond to them are the darkest.darkest.

Figure 5.9 Figure 5.9 Densitometry.Densitometry. An autoradiograph is pictured beneath a An autoradiograph is pictured beneath a densitometer scan of the same film. Notice that the areas under the densitometer scan of the same film. Notice that the areas under the three peaks of the scan are proportional to the darkness of the three peaks of the scan are proportional to the darkness of the corresponding bands on the autoradiograph.corresponding bands on the autoradiograph.

5.2.2 Phosphorimaging5.2.2 Phosphorimaging

Advantages:Advantages:

accurate in quantifying the amount of accurate in quantifying the amount of radioactivity in a substance (difference between radioactivity in a substance (difference between 10000 and 50000 dpm);10000 and 50000 dpm);

How it works :How it works :

collects radioactive emissions and analyzes them collects radioactive emissions and analyzes them

electroniclyelectronicly sample→phosphorimager plate(absorb β-rays ) sample→phosphorimager plate(absorb β-rays )

→laser scan →computerized detector→laser scan →computerized detector

Figure 5.10 False-color phosphorimager Figure 5.10 False-color phosphorimager

scan of an RNA blot.scan of an RNA blot.

After hybridizing a radioactive probe to After hybridizing a radioactive probe to

an RNA blot and washing away an RNA blot and washing away

unhybridized probe, the blot was exposed unhybridized probe, the blot was exposed

to a phosphorimager plate. The plate to a phosphorimager plate. The plate

collected energy from β-rays from the collected energy from β-rays from the

radioactive probe bound to the RNA bands, radioactive probe bound to the RNA bands,

then gave up this energy when scanned then gave up this energy when scanned

with a laser. A computer converted this with a laser. A computer converted this

energy into an image, where the colors energy into an image, where the colors

correspond to radiation intensity accordin9 correspond to radiation intensity accordin9

to the following color scale: yellow to the following color scale: yellow

(lowest) < purple < magenta < light blue < (lowest) < purple < magenta < light blue <

green < dark blue < black (highest).green < dark blue < black (highest).

5.2.3 Liquid Scintillation Counting5.2.3 Liquid Scintillation Counting

Convert the radioactive emissions Convert the radioactive emissions from a sample to photons of a visible from a sample to photons of a visible light that a photomultipllier tube can light that a photomultipllier tube can detect.detect.

Counts per minute, cpm ;Counts per minute, cpm ; 3232P is common used.P is common used.

SUMMARY SUMMARY

Detection of the tiny quantities of substances we deal Detection of the tiny quantities of substances we deal

with in molecular biology experiments generally requires with in molecular biology experiments generally requires

that we use labeled tracers. If the tracer is radioactive we that we use labeled tracers. If the tracer is radioactive we

can detect it by autoradiography, using x-ray film or a can detect it by autoradiography, using x-ray film or a

phosphorimager, or by liquid scintillation counting. phosphorimager, or by liquid scintillation counting.

5.2.4 Non-radioactive Tracers5.2.4 Non-radioactive Tracers

Significant advantage: Significant advantage:

no health hazardno health hazard Sensitivity: Sensitivity:

using multiplier effect of an enzymeusing multiplier effect of an enzyme

Figure 5.11 Detecting nucleic acids with a nonradioactive probe.Figure 5.11 Detecting nucleic acids with a nonradioactive probe.

Figure 5.11 Detecting nucleic acids with a nonradioactive probe.Figure 5.11 Detecting nucleic acids with a nonradioactive probe.

This sort of technique is usually indirect: we detect a nucleic acid of interest by This sort of technique is usually indirect: we detect a nucleic acid of interest by

hybridization to a labeled probe that can in turn be detected by virtue of its ability to hybridization to a labeled probe that can in turn be detected by virtue of its ability to

produce a colored or light-emitting substance. In this example, we execute the produce a colored or light-emitting substance. In this example, we execute the

following slaps: following slaps: (a)(a) We replicate the probe DNA in the presence of dUTP that is We replicate the probe DNA in the presence of dUTP that is

tagged with the vitamin biotin. This generates biotinylated probe DNAtagged with the vitamin biotin. This generates biotinylated probe DNA. . (b)(b) We We

denature this probe and denature this probe and (c)(c) hybridize it to the DNA we want to detect (pink). hybridize it to the DNA we want to detect (pink). (d)(d) We We

mix the hybrids with a bifunctional reagent containing both avidin and the enzyme mix the hybrids with a bifunctional reagent containing both avidin and the enzyme

alkaline phosphatase (green). The avidin binds tightly and specifically to the biotin in alkaline phosphatase (green). The avidin binds tightly and specifically to the biotin in

the probe DNA. the probe DNA. (e)(e) We add a phosphorylated compound that will become We add a phosphorylated compound that will become

chemiluminescent as soon as its phosphate group is removed. The alkaline chemiluminescent as soon as its phosphate group is removed. The alkaline

phosphatase enzymes attached to the probe cleave the phosphates from these phosphatase enzymes attached to the probe cleave the phosphates from these

substrate molecules, rendering them chemiluminescent (light-emitting). substrate molecules, rendering them chemiluminescent (light-emitting). (f)(f) We detect We detect

the light emitted from the chemiluminescent substrate with an x-ray film.the light emitted from the chemiluminescent substrate with an x-ray film.

SUMMARYSUMMARY

Some very sensitive non-radioactive labeled tracers are Some very sensitive non-radioactive labeled tracers are

now available. Those that employ chemiluminescence can now available. Those that employ chemiluminescence can

be detected by autoradiography or by phosphorimaging, just be detected by autoradiography or by phosphorimaging, just

as if they were radioactive. Those that produce colored as if they were radioactive. Those that produce colored

products can be detected directly, by observing the products can be detected directly, by observing the

appearance of colored spots. appearance of colored spots.

5.3 Using Nucleic Acid 5.3 Using Nucleic Acid HybridizationHybridization

5.3.1 5.3.1 Southern blots: Southern blots: identifying specific DNA identifying specific DNA

fragmentsfragments

Figure 5.12 Southern Figure 5.12 Southern blotting. blotting.

First, we electrophorese First, we electrophorese DNA fragments in an DNA fragments in an agarose gel. Next, we agarose gel. Next, we denature the DNA with base denature the DNA with base and transfer the single-and transfer the single-stranded DNA fragment stranded DNA fragment from the gel (yellow) to a from the gel (yellow) to a sheet of nitrocellulose (red) sheet of nitrocellulose (red) or similar material. One can or similar material. One can do this in two ways: by do this in two ways: by diffusion, in which buffer diffusion, in which buffer passes through the gel, passes through the gel, carrying the DNA with it carrying the DNA with it (left), or by electrophoresis (left), or by electrophoresis (right). Next, hybridize the (right). Next, hybridize the blot to a labeled probe and blot to a labeled probe and detect the labeled bands by detect the labeled bands by autoradiography or autoradiography or phosphorimaging. phosphorimaging.

SUMMARYSUMMARY

Labeled DNA (or RNA) probes can be used to Labeled DNA (or RNA) probes can be used to

hybridize to DNAs of the same, or very similar, sequence hybridize to DNAs of the same, or very similar, sequence

on a Southern blot. The number of bands that hybridize to a on a Southern blot. The number of bands that hybridize to a

short probe gives us an estimate of the number of closely short probe gives us an estimate of the number of closely

related genes in an organism.related genes in an organism.

5.3.2 DNA Fingerprinting 5.3.2 DNA Fingerprinting and DNA Typingand DNA Typing

Figure 5.13 DNA fingerprinting.

Figure 5.13 DNA fingerprinting. Figure 5.13 DNA fingerprinting. (a)(a) First, cut the DNA with a restriction enzyme. In this case, the First, cut the DNA with a restriction enzyme. In this case, the enzyme enzyme HaeHaeIII cuts the DNA in seven places (short arrows), III cuts the DNA in seven places (short arrows), generating eight fragments. Only three of these fragments (labeled generating eight fragments. Only three of these fragments (labeled A, B, and C according to size) contain the minisatellites, represented A, B, and C according to size) contain the minisatellites, represented by blue boxes. The other fragments (yellow) contain unrelated DNA by blue boxes. The other fragments (yellow) contain unrelated DNA sequences. sequences. (b)(b) Electrophorese the fragments from part (a), which Electrophorese the fragments from part (a), which separates them according to their sizes. All eight fragments are separates them according to their sizes. All eight fragments are present in the electrophoresis gel, but they remain invisible. The present in the electrophoresis gel, but they remain invisible. The positions of all the fragments, including the three (A, B, and C) with positions of all the fragments, including the three (A, B, and C) with minisatellites are indicated by dotted lines. minisatellites are indicated by dotted lines. (c)(c) Denature the DNA Denature the DNA fragments and Southern blot them. fragments and Southern blot them. (d)(d) Hybridize the DNA fragments Hybridize the DNA fragments on the Southern blot to a radioactive DNA with several copies of the on the Southern blot to a radioactive DNA with several copies of the minisatellite. This probe will bind to the three fragments containing minisatellite. This probe will bind to the three fragments containing the minisatellites, but with no others. Finally, use x-ray film to detect the minisatellites, but with no others. Finally, use x-ray film to detect the three labeled bands. the three labeled bands.

Figure 5.14 DNA Figure 5.14 DNA fingerprint.fingerprint.

(a) (a) The nine The nine parallel lanes parallel lanes contain DNA from contain DNA from nine unrelated nine unrelated Caucasians. Note Caucasians. Note that no two patterns that no two patterns are identical, are identical, especially at the especially at the upper end, upper end, (b)(b) The The two lanes contain two lanes contain DNA from DNA from monozygotic twins, monozygotic twins, so the patterns are so the patterns are identical.(although identical.(although there is more DNA there is more DNA in lane 10 than in in lane 10 than in lane 11). lane 11).

5.3.3 Forensic Uses of DNA 5.3.3 Forensic Uses of DNA Fingerprinting and DNA TypingFingerprinting and DNA Typing

To establish parentageTo establish parentage To identify criminalsTo identify criminals To detect heredity diseasesTo detect heredity diseases

Figure 5.15 Use of DNA typing to help identify a rapist.Figure 5.15 Use of DNA typing to help identify a rapist.

Two suspects have been accused of attacking and raping a young woman, and DNA Two suspects have been accused of attacking and raping a young woman, and DNA analyses have been performed on various samples from the suspects and the woman. Lanes analyses have been performed on various samples from the suspects and the woman. Lanes 1, 5, and 9 contain marker DNAs. Lane 2 contains DNA from the blood cells of suspect A. 1, 5, and 9 contain marker DNAs. Lane 2 contains DNA from the blood cells of suspect A. Lane 3 contains DNA from a semen sample found on the woman's clothing. Lane 4 contains Lane 3 contains DNA from a semen sample found on the woman's clothing. Lane 4 contains DNA from the blood cells of suspect B. Lane 6 contains DNA obtained by swabbing the DNA from the blood cells of suspect B. Lane 6 contains DNA obtained by swabbing the woman's vaginal canal. Lane 7 contains DNA from the woman's blood cells. Lane 8 woman's vaginal canal. Lane 7 contains DNA from the woman's blood cells. Lane 8 contains a control DNA. Lane 10 is a control containing no DNA. Partly on the basis of this contains a control DNA. Lane 10 is a control containing no DNA. Partly on the basis of this evidence, suspect B was found guilty of the crime. Note how his DNA fragments in lane 4 evidence, suspect B was found guilty of the crime. Note how his DNA fragments in lane 4 match the DNA fragments from the semen in lane 3 and the vaginal swab in lane 6.match the DNA fragments from the semen in lane 3 and the vaginal swab in lane 6.

SUMMARY SUMMARY

Modern DNA typing uses a battery of DNA probes to Modern DNA typing uses a battery of DNA probes to

detect variable sites in individual animals, including humans. detect variable sites in individual animals, including humans.

As a forensic tool, DNA typing can be used to test parentage, As a forensic tool, DNA typing can be used to test parentage,

to identify criminals, or to remove innocent people from to identify criminals, or to remove innocent people from

suspicion.suspicion.

Northern Blots:Northern Blots:Measuring Gene ActivityMeasuring Gene Activity

A Northern blot is similar to a Southern blot, but it A Northern blot is similar to a Southern blot, but it

contains electrophoretically separated RNAs instead of contains electrophoretically separated RNAs instead of

DNAs. The RNAs on the blot can be detected by DNAs. The RNAs on the blot can be detected by

hybridizing them to a labeled probe. The intensities of the hybridizing them to a labeled probe. The intensities of the

bands reveal the relative amounts of specific RNA in each.bands reveal the relative amounts of specific RNA in each.

Figure 5.16 A Northern blot. Figure 5.16 A Northern blot.

Poly(A)+ RNA was isolated from the rat tissues indicated at the top, then equal Poly(A)+ RNA was isolated from the rat tissues indicated at the top, then equal amounts of RNA from each tissue were electrophoresed and Northern blotted. amounts of RNA from each tissue were electrophoresed and Northern blotted. The RNAs on the blot were hybridized to a labeled probe for the rat The RNAs on the blot were hybridized to a labeled probe for the rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, and the blot was then glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, and the blot was then exposed to x-ray film. The bands represent the G3PDH mRNA, and their exposed to x-ray film. The bands represent the G3PDH mRNA, and their intensities are indicative of the amounts of this mRNA in each tissue. intensities are indicative of the amounts of this mRNA in each tissue.

In situ HybridizationIn situ Hybridization

Locating Genes in ChromosomesLocating Genes in Chromosomes

One can hybridize labeled probes to whole chromosomes to One can hybridize labeled probes to whole chromosomes to

locate genes or other specific DNA sequences. This type of locate genes or other specific DNA sequences. This type of

procedure is called procedure is called in situ in situ hybridization; if the probe is fluorescently hybridization; if the probe is fluorescently

labeled, the technique is called fluorescence labeled, the technique is called fluorescence in situ in situ hybridization hybridization

(FISH).(FISH).

Figure 5.17 Using a fluorescent Figure 5.17 Using a fluorescent probe to find a gene in a probe to find a gene in a chromosome by in situ chromosome by in situ

hybridization. hybridization.

A DNA probe specific for the human A DNA probe specific for the human muscle glycogen phosphorylase gene muscle glycogen phosphorylase gene was coupled to dinitrophenol. A was coupled to dinitrophenol. A human chromosome spread was human chromosome spread was then partially denatured to expose then partially denatured to expose single-stranded regions that can single-stranded regions that can hybridize to the probe. The sites hybridize to the probe. The sites where the DNP-labeled probe where the DNP-labeled probe hybridized were detected indirectly hybridized were detected indirectly as follows: A rabbit anti-DNP as follows: A rabbit anti-DNP antibody was bound to the DNP on antibody was bound to the DNP on the probe; then a goat anti-rabbitthe probe; then a goat anti-rabbit

antibody, coupled with fluoresceinantibody, coupled with fluorescein isothiocyanate (FITC), which emits yellow fluorescent isothiocyanate (FITC), which emits yellow fluorescent light, was bound to the rabbit antibody. Therefore, the chromosomal sites where the probe light, was bound to the rabbit antibody. Therefore, the chromosomal sites where the probe hybridized show up as bright yellow fluorescent spots against a red background that arises hybridized show up as bright yellow fluorescent spots against a red background that arises from staining the chromosomes with the fluorescent dye propidium iodide. This analysis from staining the chromosomes with the fluorescent dye propidium iodide. This analysis identifies chromosome 11 as the site of the glycogen phosphorylase gene. identifies chromosome 11 as the site of the glycogen phosphorylase gene.

5.3.4 DNA Sequencing5.3.4 DNA Sequencing The Sanger Chain-termination The Sanger Chain-termination

Sequencing MethodSequencing Method Maxam-Gilbert SequencingMaxam-Gilbert Sequencing

Figure 5.18 The Sanger Figure 5.18 The Sanger

dideoxy method of DNA dideoxy method of DNA

sequencing.sequencing.

Figure 5.18 The Sanger dideoxy method of DNA sequencing.Figure 5.18 The Sanger dideoxy method of DNA sequencing.

(a)(a) The primer extension (replication) reaction. A primer, 21 bases long in this case, The primer extension (replication) reaction. A primer, 21 bases long in this case, is hybridized to the single-stranded DNA to be sequenced, then mixed with the is hybridized to the single-stranded DNA to be sequenced, then mixed with the Klenow fragment of DNA polymerase and dNTPs to allow replication. One dideoxy Klenow fragment of DNA polymerase and dNTPs to allow replication. One dideoxy NTP is included to terminate replication after certain bases; in this case, ddTTP is NTP is included to terminate replication after certain bases; in this case, ddTTP is used, and it has caused termination at the second position where dTTP was called used, and it has caused termination at the second position where dTTP was called for.for.

(b)(b) Products of the four reactions (rxns). In each case, the template strand is shown Products of the four reactions (rxns). In each case, the template strand is shown at the top, with the various products underneath. Each product will begin with the at the top, with the various products underneath. Each product will begin with the 21-base primer and will have one or more nucleotides added to the 3'-end. The last 21-base primer and will have one or more nucleotides added to the 3'-end. The last nucleotide is always a dideoxy nucleotide (color) that terminated the chain. The total nucleotide is always a dideoxy nucleotide (color) that terminated the chain. The total length of each product is given in parentheses at the left end of the fragment. Thus, length of each product is given in parentheses at the left end of the fragment. Thus, we wind up with fragments ranging from 22 to 33 nucleotides long we wind up with fragments ranging from 22 to 33 nucleotides long

(c)(c) Electrophoresis of the products. The products of the four reactions are loaded into Electrophoresis of the products. The products of the four reactions are loaded into parallel lanes of a high-resolution electrophoresis gel and electrophoresed to separate parallel lanes of a high-resolution electrophoresis gel and electrophoresed to separate them according to size. By starting at the bottom and finding the shortest fragment them according to size. By starting at the bottom and finding the shortest fragment (22 bases in the A lane), then the next shortest (23 bases in the T lane), and so forth, (22 bases in the A lane), then the next shortest (23 bases in the T lane), and so forth, we can read the sequence of the product DNA. Of course, this is the complement of we can read the sequence of the product DNA. Of course, this is the complement of the template strand.the template strand.

Figure 5.19 A typical sequencing film. The sequence begins CAAAAAACGG. You can probably read the rest of the sequence to the top of the film.

SUMMARY SUMMARY

The Sanger DNA sequencing method uses dideoxy The Sanger DNA sequencing method uses dideoxy

nucleotides to terminate DNA synthesis, yielding a series nucleotides to terminate DNA synthesis, yielding a series

of DNA fragments whose sizes can be measured by of DNA fragments whose sizes can be measured by

electrophoresis. The last base in each of these fragments electrophoresis. The last base in each of these fragments

is known, since we know which dideoxy nucleotide was is known, since we know which dideoxy nucleotide was

used to terminate each reaction. Therefore, ordering these used to terminate each reaction. Therefore, ordering these

fragments by size--each fragment one (known) base fragments by size--each fragment one (known) base

longer than the next—tells us the base sequence of the longer than the next—tells us the base sequence of the

DNA.DNA.

Figure 5.20 Automated DNA sequencing.

Figure 5.20 Automated DNA sequencing. Figure 5.20 Automated DNA sequencing.

(a)(a) The primer extension reactions are run in the same way as in the manual The primer extension reactions are run in the same way as in the manual method, except that the primers in each reaction are labeled with a different method, except that the primers in each reaction are labeled with a different fluorescent molecule that emits light of a distinct color. Only one product is shown fluorescent molecule that emits light of a distinct color. Only one product is shown for each reaction, but all possible products are actually produced, just as in the for each reaction, but all possible products are actually produced, just as in the manual sequencing. manual sequencing.

(b)(b) Electrophoresis and detection of bands. The various primer extension reaction Electrophoresis and detection of bands. The various primer extension reaction products separate according to size in gel electrophoresis. The bands are color-products separate according to size in gel electrophoresis. The bands are color-coded according to the termination reaction that produced them (e.g., green for coded according to the termination reaction that produced them (e.g., green for oligonucleotides ending in ddA, blue for those ending in ddC, and so forth). A oligonucleotides ending in ddA, blue for those ending in ddC, and so forth). A laser scanner excites the fluorescent tag in each band as it passes by, and a laser scanner excites the fluorescent tag in each band as it passes by, and a detector analyzes the color of the resulting emitted light. This information is detector analyzes the color of the resulting emitted light. This information is converted to a sequence of bases and stored by a computer. converted to a sequence of bases and stored by a computer.

(c)(c) Sample printout of an automated DNA sequencing experiment. Each colored Sample printout of an automated DNA sequencing experiment. Each colored peak is a plot of the fluorescence intensity of a band as it passes through the laser peak is a plot of the fluorescence intensity of a band as it passes through the laser beam. The colors of these peaks, and those of the bands in part (b) and the tags in beam. The colors of these peaks, and those of the bands in part (b) and the tags in part (a), were chosen for convenience. They may not correspond to the actual part (a), were chosen for convenience. They may not correspond to the actual colors of the fluorescent light. colors of the fluorescent light.

5.3.5 Restriction Mapping5.3.5 Restriction Mapping

Figure 5.21 A simple restriction mapping experiment.

Figure 5.21 A simple restriction mapping experiment.Figure 5.21 A simple restriction mapping experiment.(a)(a) Determining the position of a Determining the position of a BamBamHI site. A 1.6-kb HI site. A 1.6-kb HinHindIII dIII fragment is cut by fragment is cut by BamBamHI to yield two subfragments. The sizes of HI to yield two subfragments. The sizes of these fragments are determined by electrophoresis to be 1.2 kb and these fragments are determined by electrophoresis to be 1.2 kb and 0.4kb, demonstrating that 0.4kb, demonstrating that BamBamHI cuts once, 1.2 kb from one end of HI cuts once, 1.2 kb from one end of the the HinHindIII fragment and 0.4 kb from the other end. dIII fragment and 0.4 kb from the other end. (b)(b) Determining Determining the orientation of the the orientation of the HinHindIII fragment in a cloning vector. The 1.6-dIII fragment in a cloning vector. The 1.6-kb kb HinHindIII fragment can be inserted into the dIII fragment can be inserted into the HinHindIII site of a cloning dIII site of a cloning vector, in either of two ways: vector, in either of two ways: (1)(1) with the with the BamBamHI site near an HI site near an EcoEcoRI RI site in the vector or site in the vector or (2)(2) with the with the BamBamHI site remote from an HI site remote from an EcoEcoRI RI site in the vector. To determine which, cleave the DNA with both site in the vector. To determine which, cleave the DNA with both BamBamHI and HI and EcoEcoRI and electrophorese the products to measure their RI and electrophorese the products to measure their sizes. A short fragment (0.7 kb) shows that the two sites are close sizes. A short fragment (0.7 kb) shows that the two sites are close together (left). On the other hand, a long fragment (1.5 kb) shows together (left). On the other hand, a long fragment (1.5 kb) shows that the two sites are far apart (right).that the two sites are far apart (right).

Figure 5.22 Restriction mapping of an unknown DNA.Figure 5.22 Restriction mapping of an unknown DNA. AbbreviationsAbbreviations: H=: H=HindHindIII site; P=III site; P=PstPstI site.I site.

Figure 5.23 Two potential maps of the unknown DNA.

Abbreviations: H=HindIII site; P=PstI site.

Figure 5.24 Using Southern blots in physical mapping. Figure 5.24 Using Southern blots in physical mapping.

We are mapping a 30-kb fragment is being mapping. It is cut three each by We are mapping a 30-kb fragment is being mapping. It is cut three each by EcoEcoRI (E) and RI (E) and BamBamHI (B). To aid in the mapping, first out with HI (B). To aid in the mapping, first out with EcoEcoRI, electrophorese the four resulting RI, electrophorese the four resulting fragments (fragments (EcoEcoRI-A, -B, -C, and -D), next, Southern blot the fragments and hybridize them RI-A, -B, -C, and -D), next, Southern blot the fragments and hybridize them to labeled, cloned to labeled, cloned BamBamHI-A and -B fragments. The results, shown at lower left, HI-A and -B fragments. The results, shown at lower left, demonstrate that the demonstrate that the BamBamHI-A fragment overlaps HI-A fragment overlaps EcoEcoRI-A and -C, and the RI-A and -C, and the BamBamHI-B HI-B fragment overlaps fragment overlaps EcoEcoRI-A and -D. This kind of information, coupled with digestion of RI-A and -D. This kind of information, coupled with digestion of EcoEcoRI fragments by RI fragments by BamBamH (and vice versa), allows us to piece together the whole H (and vice versa), allows us to piece together the whole restriction map. restriction map.

SummarySummary

A physical map tells us: about the spatial arrangement of A physical map tells us: about the spatial arrangement of

physical "landmarks”, such as restriction sites, on a DNA physical "landmarks”, such as restriction sites, on a DNA

molecule: One important strategy in restriction mapping (mapping molecule: One important strategy in restriction mapping (mapping

of restriction sites)is to cut the DNA in question with two or more of restriction sites)is to cut the DNA in question with two or more

restriction enzymes in separate reactions measure the Sizes of the restriction enzymes in separate reactions measure the Sizes of the

resulting fragments, then cut each with another restriction enzyme resulting fragments, then cut each with another restriction enzyme

and measure the sizes of the subfragments by get electrophoresis. and measure the sizes of the subfragments by get electrophoresis.

These sizes allow us to locate at least some of the recognition sites These sizes allow us to locate at least some of the recognition sites

relative to the others. We can improve this process considerably by relative to the others. We can improve this process considerably by

Southern blotting some of the fragments and then hybridizing these Southern blotting some of the fragments and then hybridizing these

fragments to labeled fragments generated by another restriction fragments to labeled fragments generated by another restriction

enzyme. This strategy reveals overlaps between individual enzyme. This strategy reveals overlaps between individual

restriction fragments.restriction fragments.

5.3.6 Protein Engineering5.3.6 Protein Engineering with cloned Genes: with cloned Genes:

Site-Directed MutagenesisSite-Directed Mutagenesis

Figure 5.25 PCR-based site- directed mutagenesisFigure 5.25 PCR-based site- directed mutagenesis

Figure 5.25 PCR-based site- directed mutagenesis. Figure 5.25 PCR-based site- directed mutagenesis.

We begin with a plasmid containing a gene with a TAC tyrosine codon we want We begin with a plasmid containing a gene with a TAC tyrosine codon we want

to alter to a TTC phenylalanine codon. Thus, we need to change the A-T pair to alter to a TTC phenylalanine codon. Thus, we need to change the A-T pair

(blue) in the original to a T-A pair. This plasmid was isolated from a normal strain (blue) in the original to a T-A pair. This plasmid was isolated from a normal strain

of E. coli that methylates the As of GATC sequences. The methyl group are of E. coli that methylates the As of GATC sequences. The methyl group are

indicated in yellow. indicated in yellow. (a)(a) We heat the plasmid to separate its strands. We heat the plasmid to separate its strands. (b)(b) We anneal We anneal

mutagenic primers that contain the TTC codon, or its reverse complement, GAA. mutagenic primers that contain the TTC codon, or its reverse complement, GAA.

The altered base in each primer is indicated in red. The altered base in each primer is indicated in red. (c)(c) We perform a few rounds of We perform a few rounds of

PCR (about eight) with the mutagenic primers to amplify the plasmid with its PCR (about eight) with the mutagenic primers to amplify the plasmid with its

altered codon. We use a faithful, heat-stable DNA polymerase, such as Pfu altered codon. We use a faithful, heat-stable DNA polymerase, such as Pfu

polymerase, to minimize mistakes in copying the plasmid. polymerase, to minimize mistakes in copying the plasmid. (d)(d) We treat the DNA in We treat the DNA in

the PCR reaction with the PCR reaction with DpnDpnI to digest the methylated wild-type DNA. Since the I to digest the methylated wild-type DNA. Since the

PCR product was made PCR product was made in vitroin vitro, it is not methylated and is not cut. Finally, we , it is not methylated and is not cut. Finally, we

transform E. coli cells with the treated DNA. In principle, only the mutated DNA transform E. coli cells with the treated DNA. In principle, only the mutated DNA

survives to transform. We check this by sequencing the plasmid DNA from several survives to transform. We check this by sequencing the plasmid DNA from several

clones. clones.

SummarySummary Using cloned genes, we can introduce changes at will, thus altering Using cloned genes, we can introduce changes at will, thus altering

the amino acid sequences of the protein products. The mutagenized the amino acid sequences of the protein products. The mutagenized

DNA can be made with single-stranded DNA, a mutagenic primer, DNA can be made with single-stranded DNA, a mutagenic primer,

and a standard DNA polymerase reaction, or with double-stranded and a standard DNA polymerase reaction, or with double-stranded

DNA, two complementary mutagenic primers, and PCR. Several DNA, two complementary mutagenic primers, and PCR. Several

methods are available for eliminating wild-type DNA so clones are methods are available for eliminating wild-type DNA so clones are

transformed primarily with mutagenized DNA, not with wild-type. transformed primarily with mutagenized DNA, not with wild-type.

With the PCR method, simply digesting the PCR product with With the PCR method, simply digesting the PCR product with DpnDpnI I

removes almost all of the wild-type DNA. removes almost all of the wild-type DNA.

5.4 Mapping and quantifying 5.4 Mapping and quantifying TranscriptsTranscripts

5.4.1 S1 Mapping

Figure 5.26 S1 mapping the 5’-end of a transcriptFigure 5.26 S1 mapping the 5’-end of a transcript

Figure 5.26 S1 mapping the 5’-end of a transcript. Figure 5.26 S1 mapping the 5’-end of a transcript. We begin with a cloned piece of double-stranded DNA with several known We begin with a cloned piece of double-stranded DNA with several known restriction sites. In this case, we know that the transcription start site (→) is flanked restriction sites. In this case, we know that the transcription start site (→) is flanked by two by two BamBamHI sites, and there is a single HI sites, and there is a single SalSalI site just upstream from the start site. I site just upstream from the start site. In step In step (a)(a) we cut with we cut with BamBamHI to produce the HI to produce the BamBamHI fragment shown at upper HI fragment shown at upper right. In step right. In step (b)(b) we remove the unlabeled phosphates on this fragments 5’- we remove the unlabeled phosphates on this fragments 5’-hydroxyl groups, then label these 5’-ends with polynucleotides kinase and [γ-32P] hydroxyl groups, then label these 5’-ends with polynucleotides kinase and [γ-32P] ATP. The orange circles denote the labeled ends. In step ATP. The orange circles denote the labeled ends. In step (c)(c) we cut with we cut with SalSalI and I and separatethe two resulting fragments by electrophoresis. This removes the label from separatethe two resulting fragments by electrophoresis. This removes the label from the left end of the double-stranded DNA. In step the left end of the double-stranded DNA. In step (d)(d) we denature the DNA to we denature the DNA to generate a single-stranded probe that can hybridize with the transcript (red) in step generate a single-stranded probe that can hybridize with the transcript (red) in step (e)(e). In step . In step (f)(f), we treat the hybrid with S1 nuclease. This digests the single-stranded , we treat the hybrid with S1 nuclease. This digests the single-stranded DNA on the left and the single-stranded RNA on the right of the hybrid from step DNA on the left and the single-stranded RNA on the right of the hybrid from step (e)(e). In step . In step (g)(g), we denature the remaining hybrid and electrophorese the protected , we denature the remaining hybrid and electrophorese the protected piece of the probe to see how long it is. DNA fragments of known length are piece of the probe to see how long it is. DNA fragments of known length are included as markers in a separate lane. The length of the protected probe tells us the included as markers in a separate lane. The length of the protected probe tells us the position of the transcription start site. In this case, it is 350 bp upstream of the position of the transcription start site. In this case, it is 350 bp upstream of the labeled labeled BamBamHI site in the probe. HI site in the probe.

Figure 5.27 S1 mapping the 3'-end of a transcriptFigure 5.27 S1 mapping the 3'-end of a transcript

Figure 5.27 S1 mapping the 3'-end of a transcript. Figure 5.27 S1 mapping the 3'-end of a transcript.

The principle is the same as in 5'-end mapping except that we use The principle is the same as in 5'-end mapping except that we use

a different means of labeling the probe-at its 3'-end instead of its 5'-a different means of labeling the probe-at its 3'-end instead of its 5'-

end (detailed in Figure 5,25). In step end (detailed in Figure 5,25). In step (a)(a) we cut with we cut with HinHindIII, then in dIII, then in

step step (b)(b) we label the 3'-ends of the resulting fragment. The orange we label the 3'-ends of the resulting fragment. The orange

circles denote these labeled ends. In step circles denote these labeled ends. In step (c)(c) we cut with we cut with Xho XhoI and I and

purify the left-hand labeled fragment by gel electrophoresis. In step purify the left-hand labeled fragment by gel electrophoresis. In step

(d)(d) we denature the probe and hybridize it to RNA (red) in step we denature the probe and hybridize it to RNA (red) in step (e)(e), ,

In step In step (f)(f) we remove the unprotected region of the probe (and of the we remove the unprotected region of the probe (and of the

RNA) with S1 nuclease. Finally, in step RNA) with S1 nuclease. Finally, in step (g)(g) we electrophorese the we electrophorese the

labeled protected probe to determine its size. In this case it is 225 nt labeled protected probe to determine its size. In this case it is 225 nt

long, which indicates that the 3'-end of the transcript lies 225 bp long, which indicates that the 3'-end of the transcript lies 225 bp

downstream of the labeled downstream of the labeled HinHindIII site on the left-hand end of the dIII site on the left-hand end of the

probe.probe.

Figure 5.28 3'-end labeling a DNA by end-filling.

The DNA fragment at the top has been created by cutting with HindIII, which

leaves 5'-overhangs at each end, as shown. These can be filled in with a fragment of

DNA polymerase called the Klenow fragment. This enzyme fragment has an

advantage over the whole DNA polymerase in that it lacks the normal 5' →3'

exonuclease activity, which could degrade the 5'-overhangs before they could be

filled in. We run the end-filling reaction with all four nucleotides, one of which

(dATP) is labeled, so the DNA end will become labeled. If we want to incorporate

more label into the end, we can include more than one labeled nucleotide.

SUMMARYSUMMARY In S1 mapping we use a labeled DNA probe to detect the 5'- or In S1 mapping we use a labeled DNA probe to detect the 5'- or

3'-end of a transcript. Hybridization of the probe to the transcript 3'-end of a transcript. Hybridization of the probe to the transcript

protects a portion of the probe from digestion by S1 nuclease, which protects a portion of the probe from digestion by S1 nuclease, which

specifically degrades single-stranded polynucleotides. The length of specifically degrades single-stranded polynucleotides. The length of

the section of probe protected by the transcript locates the end of the the section of probe protected by the transcript locates the end of the

transcript, relative to the known location of an end of the probe. transcript, relative to the known location of an end of the probe.

Since the amount of probe protected by the transcript is proportional Since the amount of probe protected by the transcript is proportional

to the concentration of transcript, $1 mapping can also be used as a to the concentration of transcript, $1 mapping can also be used as a

quantitative method. RNase mapping is a variation on S1 mapping quantitative method. RNase mapping is a variation on S1 mapping

that uses an RNA probe and RNase instead of a DNA probe and S1 that uses an RNA probe and RNase instead of a DNA probe and S1

nuclease.nuclease.

5.4.2 Primer Extension5.4.2 Primer Extension

Figure 5.29 Figure 5.29

Primer extension.Primer extension.

Figure 5.29 Primer extension. Figure 5.29 Primer extension.

(a)(a) Transcription occurs naturally within the cell, so we do not have to perform this Transcription occurs naturally within the cell, so we do not have to perform this

step; we just harvest cellular RNAstep; we just harvest cellular RNA. (b). (b) Knowing the sequence of at least part of the Knowing the sequence of at least part of the

transcript, we synthesize and label a DNA oligonucleotide that is complementary to a transcript, we synthesize and label a DNA oligonucleotide that is complementary to a

region not too far from the suspected 5‘-end, then we hybridize this oligonucleotide to region not too far from the suspected 5‘-end, then we hybridize this oligonucleotide to

the transcript. It should hybridize specifically to this transcript and to no others. the transcript. It should hybridize specifically to this transcript and to no others. (c)(c) We We

use reverse transcriptase to extend the primer by synthesizing DNA complementary to use reverse transcriptase to extend the primer by synthesizing DNA complementary to

the transcript, up to its 5’-end. If the primer itself is not labeled, or if we want to the transcript, up to its 5’-end. If the primer itself is not labeled, or if we want to

introduce extra label into the extended primer, we can include labeled nucleotides in introduce extra label into the extended primer, we can include labeled nucleotides in

this step.this step. (d)(d) We denature the hybrid and electrophorese the labeled, extended primer. We denature the hybrid and electrophorese the labeled, extended primer.

In separate lanes we run sequencing reactions, performed with the same primer, as In separate lanes we run sequencing reactions, performed with the same primer, as

markers. In principle, this can tell us the transcription start site to the exact base. In this markers. In principle, this can tell us the transcription start site to the exact base. In this

case, the extended primer (arrow) co-electrophoreses with a DNA fragment in the case, the extended primer (arrow) co-electrophoreses with a DNA fragment in the

sequencing A lane. Since the same primer was used in the primer extension reaction sequencing A lane. Since the same primer was used in the primer extension reaction

and in all the sequencing reactions, this tells us that the 5'-end of this transcript and in all the sequencing reactions, this tells us that the 5'-end of this transcript

corresponds to the middle A (underlined) in the sequence TTCGACTGACAGT.corresponds to the middle A (underlined) in the sequence TTCGACTGACAGT.

SUMMARYSUMMARY Using primer extension we can locate the 5'-end of a transcript Using primer extension we can locate the 5'-end of a transcript

by hybridizing an oligonucleotide primer to the RNA of interest, by hybridizing an oligonucleotide primer to the RNA of interest,

extending the primer with reverse transcriptase to the 5'-end of the extending the primer with reverse transcriptase to the 5'-end of the

transcript, and electrophoresing the reverse transcript to determine its transcript, and electrophoresing the reverse transcript to determine its

size. The intensity of the signal obtained by this method is a measure size. The intensity of the signal obtained by this method is a measure

of the concentration of the transcript.of the concentration of the transcript.

5.4.3 Run-off Transcription and 5.4.3 Run-off Transcription and G-Less Cassette TranscriptionG-Less Cassette Transcription

Whether transcription initiates in the right Whether transcription initiates in the right place?place?How much of this accurate transcription How much of this accurate transcription occurredoccurred

SUMMARYSUMMARY

Using primer extension we can locate the 5'-end of a Using primer extension we can locate the 5'-end of a

transcript by hybridizing an oligonucleotide primer to the RNA transcript by hybridizing an oligonucleotide primer to the RNA

of interest, extending the primer with reverse transcriptase to of interest, extending the primer with reverse transcriptase to

the 5'-end of the transcript, and electrophoresing the reverse the 5'-end of the transcript, and electrophoresing the reverse

transcript to determine its size. The intensity of the signal transcript to determine its size. The intensity of the signal

obtained by this method is a measure of the concentration of the obtained by this method is a measure of the concentration of the

transcript.transcript.

can be measured by gel electrophoresis and corresponds to the distance between the can be measured by gel electrophoresis and corresponds to the distance between the start of transcription and the known restriction site at the 3’-end of the shortened gene start of transcription and the known restriction site at the 3’-end of the shortened gene (a SmaI site in this case). The more actively this gene is transcribed, the stronger the (a SmaI site in this case). The more actively this gene is transcribed, the stronger the 327- nucleotides signal will be. 327- nucleotides signal will be.

Figure 5.30 Run-off Figure 5.30 Run-off transcription. transcription.

We begin by cutting We begin by cutting the cloned gene, whose the cloned gene, whose transcription we want to transcription we want to assay, with a restriction assay, with a restriction enzyme. We then enzyme. We then transcribe this truncated transcribe this truncated gene in vitro. When the gene in vitro. When the RNA polymerase RNA polymerase (orange) reaches the end (orange) reaches the end of the shortened gene, it of the shortened gene, it falls off and releases the falls off and releases the run-off transcript (red). run-off transcript (red). The size of the run-off The size of the run-off transcript (327 transcript (327 nucleotides in this case)nucleotides in this case)

G-less cassette assay. G-less cassette assay.

Figure 5.31 G-less cassette assay. Figure 5.31 G-less cassette assay. (a)(a) Transcribe a template with a G-less cassette Transcribe a template with a G-less cassette (pink) inserted downstream of the promoter in vitro in the absence of GTP. This (pink) inserted downstream of the promoter in vitro in the absence of GTP. This cassette is 355 bp long, contains no G’s in the nontemplate strand, and is followed cassette is 355 bp long, contains no G’s in the nontemplate strand, and is followed by the sequence TGC, so transcription stops just before the G, producing a by the sequence TGC, so transcription stops just before the G, producing a transcript 356 nt long. transcript 356 nt long. (b)(b) Electrophorese the labeled transcript and autoradiograph Electrophorese the labeled transcript and autoradiograph the gel and measure the intensity of the signal, which indicates how actively the the gel and measure the intensity of the signal, which indicates how actively the cassette was transcribed. cassette was transcribed.

5.5 Measuring Transcription Rates5.5 Measuring Transcription Rates

in vivo in vivo

Nuclear Run-on TranscriptionNuclear Run-on TranscriptionReporter Gene TranscriptionReporter Gene TranscriptionMeasuring protein accumulation in vivoMeasuring protein accumulation in vivo

SUMMARY SUMMARY

Nuclear run-on transcription is a way of Nuclear run-on transcription is a way of

ascertaining which genes are active in a given cell by ascertaining which genes are active in a given cell by

allowing transcription of these genes to continue in allowing transcription of these genes to continue in

isolated nuclei. Specific transcripts can be identified isolated nuclei. Specific transcripts can be identified

by their hybridization to known DNAs on dot blots. by their hybridization to known DNAs on dot blots.

We can also use the run-on assay to determine the We can also use the run-on assay to determine the

effects of assay conditions on nuclear transcription.effects of assay conditions on nuclear transcription.

stranded DNA from genes stranded DNA from genes XX, , YY, and , and ZZ on nitrocellulose, or another suitable medium, and on nitrocellulose, or another suitable medium, and hybridize the blot to the labeled run-on transcripts. Since gene hybridize the blot to the labeled run-on transcripts. Since gene YY was transcribed in the was transcribed in the run-on reaction, it will be labeled, and the gene run-on reaction, it will be labeled, and the gene YY spot will become labeled. On the other spot will become labeled. On the other hand, since genes hand, since genes XX and and ZZ were not active, no labeled were not active, no labeled XX and and ZZ transcripts were made, so the transcripts were made, so the XX and and ZZ spots remain unlabeled. spots remain unlabeled.

Figure 5.32 Nuclear run-on Figure 5.32 Nuclear run-on transcription. transcription. (a)(a) The run-on The run-on reaction. We start with cells reaction. We start with cells that are in the process of that are in the process of transcribing thetranscribing the Y Y gene, but not gene, but not the the XX or or ZZ genes. The RNA genes. The RNA polymerase (orange) is making polymerase (orange) is making a transcript (blue) of the a transcript (blue) of the YY gene. gene. We isolate nuclei from these We isolate nuclei from these cells and incubate them with cells and incubate them with nucleotides so transcription can nucleotides so transcription can continue (run-on). We also continue (run-on). We also include a labeled nucleotide in include a labeled nucleotide in the run-on reaction so the the run-on reaction so the transcript will become labeled transcript will become labeled (red). Finally, we extract the (red). Finally, we extract the labeled run-on transcripts. labeled run-on transcripts. (b)(b) Dot blot assay. We spot single-Dot blot assay. We spot single-

SUMMARYSUMMARY

To measure the activity of a promoter, we can link it to a To measure the activity of a promoter, we can link it to a

reporter gene, such as the genes encoding β-galactosidase, reporter gene, such as the genes encoding β-galactosidase,

CAT, or luciferase, and let the easily assayed reporter gene CAT, or luciferase, and let the easily assayed reporter gene

products tell us indirectly the activity of the promoter. We products tell us indirectly the activity of the promoter. We

can also use reporter genes to detect changes in translational can also use reporter genes to detect changes in translational

efficiency after we alter regions of a gene that effect efficiency after we alter regions of a gene that effect

translation.translation.

Using a report gene

Figure 5.33 Using a reporter gene. (a) Outline of the method. Step 1: We start with a plasmid containing gene (X, blue) under control of its own promoter (yellow) and use restriction enzymes to remove the coding region of gene X. Step 2: We insert the bacterial cat gene under control of the X gene's promoter. Step 3: We insert this construct into eukaryotic cells Step 4: After a period of time, we make an extract of the cells. Step 5: To begin our CAT assay, we add 14C-CAM and the acetyl donor acetyl CoA. Step 6: We perform thin-layer chromatography to separate acetylated and unacetylated species of CAM. Step 7: Finally, we subject the thin layer to autoradiography to visualize CAM and its acetylated derivatives. Here we see CAM near the bottom of the autoradiogram and two acetylated forms of CAM, with higher mobility, near the top. (b) Actual experimental results. Again, the parent CAM is near the bottom, and two acetylated forms of CAM are nearer the top. The experimenters scraped these radioactive species off of the thin layer plate and subjected them to liquid scintillation counting, yielding the CAT activity values reported at the bottom (averages of duplicate lanes) Lane 1 is a negative control with no cell extract.

Measuring protein accumulation in vivoMeasuring protein accumulation in vivo

Immunobloting, western blotingImmunobloting, western bloting Immunoprecipitation Immunoprecipitation

Summary Summary

Gene expression can be quantified by Gene expression can be quantified by measuring the accumulation of the protein measuring the accumulation of the protein products of genes. Immunoblotting and products of genes. Immunoblotting and immunoprecipitation are the favorite ways immunoprecipitation are the favorite ways of accomplishing this task. of accomplishing this task.

5.6 5.6 Assaying DNA-Protein Assaying DNA-Protein InteractionsInteractions

5.5.1 Filter binding 5.5.1 Filter binding

5.5.2 Gel Mobility Shift5.5.2 Gel Mobility Shift

5.5.3 DNase Footprinting5.5.3 DNase Footprinting

5.5.4 DMS Footprinting5.5.4 DMS Footprinting

scintillation counting. None of the radioactivity slicks to the filler, indicating that double-scintillation counting. None of the radioactivity slicks to the filler, indicating that double-stranded DNA does not bind to nitrocellulose, Single-stranded DNA. on the other hand, stranded DNA does not bind to nitrocellulose, Single-stranded DNA. on the other hand, binds tightly. binds tightly. (b)(b) ProteinProtein. We label a protein (green) and filter it through nitrocellulose. The . We label a protein (green) and filter it through nitrocellulose. The protein binds to the nitrocellulose. protein binds to the nitrocellulose. (c)(c) Double-stranded DNA-protein complexDouble-stranded DNA-protein complex. We mix an . We mix an end-labeled double-stranded DNA (red) with an unlabeled protein (green) to which it binds end-labeled double-stranded DNA (red) with an unlabeled protein (green) to which it binds to form a DNA-protein complex. Then we filter the complex through nitrocellulose. The to form a DNA-protein complex. Then we filter the complex through nitrocellulose. The labeled DNA now binds to the filter because of its association with the protein Thus, double-labeled DNA now binds to the filter because of its association with the protein Thus, double-stranded DNA-protein complexes bind to nitrocellulose, and this provides a convenient stranded DNA-protein complexes bind to nitrocellulose, and this provides a convenient assay for association between DNA and protein.assay for association between DNA and protein.

Figure 5.34 Figure 5.34 Nitrocellulose filter Nitrocellulose filter

binding assay. binding assay.

(a)(a) Double-stranded Double-stranded DNADNA . We end-label . We end-label double-stranded double-stranded DNA (red) and pass DNA (red) and pass it through a it through a nitrocellulose filter. nitrocellulose filter. Then we monitor Then we monitor the radioactivity on the radioactivity on the filter and in the the filter and in the filtrate by liquidfiltrate by liquid

SUMMARYSUMMARY

Filter binding as a means of measuring DNA-protein Filter binding as a means of measuring DNA-protein

interaction is based on the fact that double-stranded DNA will not interaction is based on the fact that double-stranded DNA will not

bind by itself to a nitrocellulose filter, or similar medium, but a bind by itself to a nitrocellulose filter, or similar medium, but a

protein-DNA complex wilt. Thus, we can label a double-stranded protein-DNA complex wilt. Thus, we can label a double-stranded

DNA, mix it with a protein, and assay protein-DNA binding by DNA, mix it with a protein, and assay protein-DNA binding by

measuring the amount of label retained by the filter.measuring the amount of label retained by the filter.

Figure 5.35 Gel mobility shift assay. We subject pure, labeled DNA or DNA-protein complexes to gel electrophoresis, then autoradiograph the gel to detect the DNAs and complexes. Lane 1 shows the high mobility of bare DNA. Lane 2 shows the mobility shift that occurs upon binding a protein (red) to the DNA. Lane 3 shows the supershift caused by binding a second protein (yellow) to the DNA-protein complex. The orange dots at the ends of the DNAs represent terminal labels.

SUMMARYSUMMARY

A gel mobility shift assay detects interaction between a A gel mobility shift assay detects interaction between a

protein and DNA by the retardation of the electrophoretic protein and DNA by the retardation of the electrophoretic

mobility of a small DNA that occurs upon binding to a proteinmobility of a small DNA that occurs upon binding to a protein.

Figure 5.36 DNase footprinting.

Figure 5.36 DNase footprinting. Figure 5.36 DNase footprinting. (a)(a) Outline of method.Outline of method. We begin with a double-stranded DNA, labeled at We begin with a double-stranded DNA, labeled at one end (orange). Next, we bind a protein to the DNA. Next, we digest the one end (orange). Next, we bind a protein to the DNA. Next, we digest the DNA-protein complex under mild conditions with DNase I, so as to DNA-protein complex under mild conditions with DNase I, so as to introduce approximately one break per DNA molecule. Next, we remove introduce approximately one break per DNA molecule. Next, we remove the protein and denature the DNA, yielding the end-labeled fragments the protein and denature the DNA, yielding the end-labeled fragments shown at center. Notice that the DNase cut the DNA at regular intervals shown at center. Notice that the DNase cut the DNA at regular intervals except where the protein bound and protected the DNA. Finally, except where the protein bound and protected the DNA. Finally, weelectrophorese the labeled fragments and perform autoradiography to weelectrophorese the labeled fragments and perform autoradiography to detect them. The three lanes represent DNA that was bound to 0, 1, and 5 detect them. The three lanes represent DNA that was bound to 0, 1, and 5 units of proteins. The lane with no protein shows a regular ladder of units of proteins. The lane with no protein shows a regular ladder of fragments. The lane with one unit of protein shows some protection, and fragments. The lane with one unit of protein shows some protection, and the lane with 5 units of proteins shows complete protection in the middle. the lane with 5 units of proteins shows complete protection in the middle. This protected area is called the footprint; it shows us where the protein This protected area is called the footprint; it shows us where the protein bound to the DNA. We usually include sequencing reactions performed on bound to the DNA. We usually include sequencing reactions performed on the same DNA in parallel lanes. These serve as size markers, so we can tell the same DNA in parallel lanes. These serve as size markers, so we can tell exactly where the protein bound. exactly where the protein bound. (b) Actual experimental results(b) Actual experimental results. Lanes 1-4 contained DNA bound to 0, 10, . Lanes 1-4 contained DNA bound to 0, 10, 18, and 90 picomoles (pmol) of protein, respectively (1 pmol-1018, and 90 picomoles (pmol) of protein, respectively (1 pmol-10 -12-12mol). mol).

Figure 5.37 DMS footprinting.Figure 5.37 DMS footprinting.

Figure 5.37 DMS footprinting. Figure 5.37 DMS footprinting.

(a)(a) Outline of the method. As in DNase footprinting, we start with an end- Outline of the method. As in DNase footprinting, we start with an end-labeled DNA, then bind a protein (yellow) to it. In this case, the protein causes labeled DNA, then bind a protein (yellow) to it. In this case, the protein causes some tendency of the DNA duplex to melt in one region, represented by the some tendency of the DNA duplex to melt in one region, represented by the small “bubble”. Next, we methylate the DNA with DMS. This adds methyl small “bubble”. Next, we methylate the DNA with DMS. This adds methyl groups (CH3, red) to certain bases in the DNA. We do this under mild groups (CH3, red) to certain bases in the DNA. We do this under mild condition so that, on average, only one methylated base occurs per DNA condition so that, on average, only one methylated base occurs per DNA molecule (even though all seven methylations are shown together on one strand molecule (even though all seven methylations are shown together on one strand for convenience here). Next, we use the Maxam-Gilbert sequencing reagents to for convenience here). Next, we use the Maxam-Gilbert sequencing reagents to remove methylated purines from the DNA, then to break the DNA at these remove methylated purines from the DNA, then to break the DNA at these apurinic sites. This yields the labeled DNA fragments depicted at center. We apurinic sites. This yields the labeled DNA fragments depicted at center. We electrophorese these fragments and autoradiograph the gel to give the results electrophorese these fragments and autoradiograph the gel to give the results shown at bottom. Notice that three sites are protected against methylation by shown at bottom. Notice that three sites are protected against methylation by the protein, but one site is actually made more sensitive to methylation (darker the protein, but one site is actually made more sensitive to methylation (darker band). This is because of the opening up of the double helix that occurs in this band). This is because of the opening up of the double helix that occurs in this position. position.

(b)(b) Actual experimental results. Lanes 1 and 4 have no added protein, while Actual experimental results. Lanes 1 and 4 have no added protein, while lanes 2 and 3 have increasing concentrations of a protein that binds to this lanes 2 and 3 have increasing concentrations of a protein that binds to this region of the DNA. The bracket indicates a pronounced footprint region. The region of the DNA. The bracket indicates a pronounced footprint region. The asterisks denote bases made more susceptible to methylation by protein asterisks denote bases made more susceptible to methylation by protein binding. binding.

SUMMARY SUMMARY

Footprinting is a means of finding the target DNA sequence, or Footprinting is a means of finding the target DNA sequence, or

binding site, of a DNA-binding protein. We perform DNase binding site, of a DNA-binding protein. We perform DNase

footprinting by binding the protein to its end-labeled DNA target, footprinting by binding the protein to its end-labeled DNA target,

then attacking the DNA-protein complex with DNase. When we then attacking the DNA-protein complex with DNase. When we

electrophorese the resulting DNA fragments, the protein binding electrophorese the resulting DNA fragments, the protein binding

site shows up as a gap, or "footprint" in the pattern where the site shows up as a gap, or "footprint" in the pattern where the

protein protected the DNA from degradation. DMS foorprinting protein protected the DNA from degradation. DMS foorprinting

follows a similar principle, except that we use the DNA follows a similar principle, except that we use the DNA

methylating agent DMS, instead of DNase, to attack the DNA-methylating agent DMS, instead of DNase, to attack the DNA-

protein complex. The DNA is then broken at the methylated sites. protein complex. The DNA is then broken at the methylated sites.

Unmethylated (or hypermethylated) sites show up upon Unmethylated (or hypermethylated) sites show up upon

electrophoresis of the labeled DNA fragments-and demonstrate electrophoresis of the labeled DNA fragments-and demonstrate

where the protein bound to the DNA.where the protein bound to the DNA.

5.7 Knockouts5.7 Knockouts

SummarySummary

To probe the role of a gene, molecular biologists can To probe the role of a gene, molecular biologists can

perform targeted disruption of the corresponding gene in perform targeted disruption of the corresponding gene in

a mouse, and then observe the effects of that mutation on a mouse, and then observe the effects of that mutation on

the “knockout mouse”.the “knockout mouse”.

1.1. We start with a plasmid containing the gene we want to We start with a plasmid containing the gene we want to inactivate (the target gene, green) plus a thymidine inactivate (the target gene, green) plus a thymidine kinase gene (kinase gene (tktk). We interrupt the target gene by splicing ). We interrupt the target gene by splicing the neomycin-resistance gene (red) into it. the neomycin-resistance gene (red) into it.

2.2. We collect stem cells (tan) from a brown mouse We collect stem cells (tan) from a brown mouse embryo.embryo.

Making a knockout mouseMaking a knockout mouse ：：Stage 1Stage 1 creating stem cells with an interrupted genecreating stem cells with an interrupted gene

3. We transfect these cells with the plasmid containing the interrupted target 3. We transfect these cells with the plasmid containing the interrupted target

gene. gene.

4 and 5. Three kinds of products result from this transfection: 4a. Homologous 4 and 5. Three kinds of products result from this transfection: 4a. Homologous

recombination between the interrupted target gene in the plasmid and the recombination between the interrupted target gene in the plasmid and the

homologous, wild-type gene causes replacement of the wild-type gene in the homologous, wild-type gene causes replacement of the wild-type gene in the

cellular genome by the interrupted gene (5a). 4b. Nonspecific recombination cellular genome by the interrupted gene (5a). 4b. Nonspecific recombination

with a nonhornologous sequence in the cellular genome results in random with a nonhornologous sequence in the cellular genome results in random

insertion of the interrupted target gene plus the tk gene into the cellular insertion of the interrupted target gene plus the tk gene into the cellular

genome (5b). 4c. When no recombination occurs, the interrupted target gene genome (5b). 4c. When no recombination occurs, the interrupted target gene

is not integrated into the cellular genome at all (5c). is not integrated into the cellular genome at all (5c).

6. The cells resulting from these three events are color coded as indicated: 6. The cells resulting from these three events are color coded as indicated:

Homologous recombination yields a cell (red) with an interrupted target gene Homologous recombination yields a cell (red) with an interrupted target gene

(6a); nonspecific recombination yields a cell (blue) with the interrupted target (6a); nonspecific recombination yields a cell (blue) with the interrupted target

gene and the tk gene inserted at random (6b); no ecombination yields a cell gene and the tk gene inserted at random (6b); no ecombination yields a cell

(tan) with no integration of the interrupted gene,(tan) with no integration of the interrupted gene,

Making a knockout mouseMaking a knockout mouse: Stage 2Stage 2 placing the interrupted gene in the animalplacing the interrupted gene in the animal

(1) We inject the ceils with the interrupted gene (see stage 1) into a blastocyst-stage embryo from black parent mice

(2) We transplant this mixed embryo to the uterus of a surrogate mother. (3) The surrogate mother gives birth to a chimeric mouse, which we can

identify by its black arid brown coat. (Recall that the altered cells came from an agouti [brown] mouse, and they were placed into an embryo from a black mouse.)

(4) We allow the chimeric mouse (a male) to mature.

(5) We mate it with a wild-type black female We can discard (5) We mate it with a wild-type black female We can discard any black offspring, which must have derived from the wild-any black offspring, which must have derived from the wild-type blastocyst; only brown mice could have derived from type blastocyst; only brown mice could have derived from the transplanted cells.the transplanted cells.

(6) We select a brown brother and sister pair, both of which show evidence of an (6) We select a brown brother and sister pair, both of which show evidence of an

interrupted target gene (by Southern blot analysis), and mate them Again we interrupted target gene (by Southern blot analysis), and mate them Again we

examine the DNA of the brown progeny by Southern blotting. This time, we find examine the DNA of the brown progeny by Southern blotting. This time, we find

one animal that is homozygous for the interrupted target gene This is our one animal that is homozygous for the interrupted target gene This is our

knockout mouse We can now observe this animal to determine the effects of knockout mouse We can now observe this animal to determine the effects of

knocking Out the target gene.knocking Out the target gene.

Biotechnology: Present and Future