UNIT 4 Techniques used in Molecular Biology. Objectives On completion of this unit students will be...

UNIT 4UNIT 4

Techniques used Techniques used in Molecular in Molecular

BiologyBiology

ObjectivesObjectives

On completion of this unit students will be On completion of this unit students will be able to:able to:

Outline the steps in Polymerase Chain ReactionOutline the steps in Polymerase Chain Reaction Analyse DNA agarose gel electrophoretogramsAnalyse DNA agarose gel electrophoretograms Describe DNA hybridization and its application Describe DNA hybridization and its application

in probe synthesisin probe synthesis Differentiate between Northern, Southern and Differentiate between Northern, Southern and

Western BlotsWestern Blots Outline the methods of DNA sequencingOutline the methods of DNA sequencing

Polymerase Chain Polymerase Chain ReactionReaction

The PCR technique is basically a primer The PCR technique is basically a primer extension reaction for amplifying specific extension reaction for amplifying specific nucleic acids nucleic acids in vitroin vitro..

PCR will allow a short stretch of DNA (usually PCR will allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, million fold so that one can determine its size, nucleotide sequence, etc. nucleotide sequence, etc.

The particular stretch of DNA to be amplified The particular stretch of DNA to be amplified is called the target sequenceis called the target sequence

The target sequence is identified by a specific The target sequence is identified by a specific pair of DNA primers (oligonucleotides) usually pair of DNA primers (oligonucleotides) usually about 20 nucleotides in lengthabout 20 nucleotides in length. .

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Primers must be duplicates of nucleotide Primers must be duplicates of nucleotide sequences on either side of the piece of sequences on either side of the piece of DNA of interest.DNA of interest.

The exact order of the primers' The exact order of the primers' nucleotides must already be known. nucleotides must already be known.

Primers can be constructed in the lab, or Primers can be constructed in the lab, or purchased from commercial suppliers.purchased from commercial suppliers.


The cycling reactionsThe cycling reactions : :

There are three major steps in a PCR, There are three major steps in a PCR, which are repeated for 30 or 40 cycles. which are repeated for 30 or 40 cycles.

This is done on an automated cycler, which This is done on an automated cycler, which can heat and cool the tubes with the can heat and cool the tubes with the reaction mixture in a very short time. reaction mixture in a very short time.

ThermocyclerThermocycler

Steps in PCRSteps in PCR

Three steps in PCRThree steps in PCR


Because both strands are copied during Because both strands are copied during PCR, there is an exponential increase of PCR, there is an exponential increase of the number of copies of the gene. the number of copies of the gene.

Suppose there is only one copy of the Suppose there is only one copy of the wanted gene before the cycling starts, wanted gene before the cycling starts, after one cycle, there will be 2 copies, after after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three two cycles, there will be 4 copies, three cycles will result in 8 copies and so on. cycles will result in 8 copies and so on.

DenaturationDenaturation

94°C 94°C

During the denaturation, the double During the denaturation, the double strand melts open to single stranded strand melts open to single stranded DNA.DNA.

All enzymatic reactions stop (for All enzymatic reactions stop (for example : the extension from a previous example : the extension from a previous cycle). cycle).

Annealing/ HybridizationAnnealing/ Hybridization54°C54°C : :

hydrogen bonds are constantly formed and broken hydrogen bonds are constantly formed and broken between the single stranded primer and the single between the single stranded primer and the single stranded template. stranded template.

The more stable bonds last a little bit longer The more stable bonds last a little bit longer (primers that fit exactly).(primers that fit exactly).

The polymerase can attach to pieces of double The polymerase can attach to pieces of double stranded DNA (template and primer), and starts stranded DNA (template and primer), and starts copying the template. copying the template.

Once there are a few bases built in, the hydrogen Once there are a few bases built in, the hydrogen bond is so strong between the template and the bond is so strong between the template and the primer, that it does not break anymore. primer, that it does not break anymore.

Extension /ElongationExtension /Elongation

72°C72°C : :

This is the ideal working temperature for the This is the ideal working temperature for the polymerase. polymerase.

Primers that are on positions with no exact match, Primers that are on positions with no exact match, get loose again (because of the higher temperature) get loose again (because of the higher temperature) and don't give an extension of the fragment.and don't give an extension of the fragment.

The bases (complementary to the template) are The bases (complementary to the template) are coupled to the primer on the 3' side (the coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3'). polymerase adds dNTP's from 5' to 3').


The use of a thermostable polymerase The use of a thermostable polymerase allows:allows:

The dissociation of newly formed The dissociation of newly formed complimentary DNAcomplimentary DNA

Subsequent annealing or hybridization of Subsequent annealing or hybridization of primers to the target sequence with primers to the target sequence with minimal loss of enzymatic activity. minimal loss of enzymatic activity.


Is there a gene copied during PCR and is it Is there a gene copied during PCR and is it the the

right size? right size?

Before the PCR product is used in further Before the PCR product is used in further applications, it has to be checked if there is a applications, it has to be checked if there is a product formed. product formed.

Factors that affect yield:Factors that affect yield:

quality of the DNA is poorquality of the DNA is poor one of the primers doesn't fitone of the primers doesn't fit too much starting template.too much starting template.


The product is of the right size:The product is of the right size:

It is possible that there is a product, for example a It is possible that there is a product, for example a band of 500 bases, but the expected gene should be band of 500 bases, but the expected gene should be 1800 bases long. 1800 bases long.

Factors that affect specificity:Factors that affect specificity:

one of the primers probably fits on a part of the one of the primers probably fits on a part of the gene closer to the other primer. gene closer to the other primer.

It is also possible that both primers fit on a totally It is also possible that both primers fit on a totally different gene. different gene.

Uses of PCRUses of PCR

1. The method is especially useful for searching out disease organisms that are difficult or impossible to culture:

such as many kinds of bacteria, fungi, and such as many kinds of bacteria, fungi, and viruses, because it can generate analyzable viruses, because it can generate analyzable quantities of the organism's genetic material quantities of the organism's genetic material for identification. for identification.

Uses of PCRUses of PCR2.2.PCR can also be more accurate than standard tests. PCR can also be more accurate than standard tests.

The technique is used to detect bacterial infections by The technique is used to detect bacterial infections by detecting their DNAdetecting their DNA bacterial ear infection. Sensitive even when culture methods bacterial ear infection. Sensitive even when culture methods

failed to detect it. failed to detect it. lyme disease, the painful joint inflammation caused by bacteria lyme disease, the painful joint inflammation caused by bacteria

transmitted through tick bites., is usually diagnosed on the basis transmitted through tick bites., is usually diagnosed on the basis of symptom patterns. PCR can be used to identify the organism's of symptom patterns. PCR can be used to identify the organism's DNA permitting speedy treatment that can prevent serious DNA permitting speedy treatment that can prevent serious complications.complications.

PCR is the most sensitive and specific test for PCR is the most sensitive and specific test for Helicobacter Helicobacter pyloripylori, the disease organism now known to cause almost all , the disease organism now known to cause almost all stomach ulcers. stomach ulcers.

It can detect the AIDS virus sooner during the first few weeks It can detect the AIDS virus sooner during the first few weeks after infection than the standard ELISA test. PCR looks directly after infection than the standard ELISA test. PCR looks directly for the virus‘ unique nucleic acid, instead of the method for the virus‘ unique nucleic acid, instead of the method employed by the standard test, which looks for indirect evidence employed by the standard test, which looks for indirect evidence that the virus is present by searching for antibodies the body has that the virus is present by searching for antibodies the body has made against it..made against it..

Uses of PCRUses of PCR

3.3. The method is also leading to new The method is also leading to new kinds of genetic testing.kinds of genetic testing.

These tests diagnose not only people with These tests diagnose not only people with inherited disorders, but also people who carry inherited disorders, but also people who carry deleterious mutations that could be passed to deleterious mutations that could be passed to their children. their children.


4.4. PCR can provide enormous peace of mind to PCR can provide enormous peace of mind to people who are trying to have children-people who are trying to have children-

for example, by reassuring anxious parents-to-be that for example, by reassuring anxious parents-to-be that they run no risk of having a child with a particular they run no risk of having a child with a particular genetic disease. genetic disease.

The technique even saves the lives of babies before The technique even saves the lives of babies before they are born: detect whether the blood groups of they are born: detect whether the blood groups of mother and fetus are incompatible. This condition often mother and fetus are incompatible. This condition often leads to severe disability and even death of the fetus, leads to severe disability and even death of the fetus, but can be treated successfully in the womb with but can be treated successfully in the womb with enough advance warning-thanks to PCR.enough advance warning-thanks to PCR.


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Gel electrophoresisGel electrophoresis

a method that separates macromolecules-a method that separates macromolecules-either nucleic acids or proteins-on the basis either nucleic acids or proteins-on the basis of:of:

sizesize

electric chargeelectric charge

other physical properties, such as topology.other physical properties, such as topology.

Pouring a gelPouring a gel

Loading a gelLoading a gel

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Gel electrophoresisGel electrophoresis

A gel is a colloid in a solid form. A gel is a colloid in a solid form.

The term electrophoresis describes the The term electrophoresis describes the migration of charged particle under the migration of charged particle under the influence of an electric field. influence of an electric field.

ElectroElectro refers to the energy of electricity. refers to the energy of electricity.

PhoresisPhoresis, from the Greek verb , from the Greek verb phorosphoros, means , means "to carry across." "to carry across."

Gel ElectrophoresisGel Electrophoresis Thus, gel electrophoresis refers to the Thus, gel electrophoresis refers to the

technique in which molecules are forced technique in which molecules are forced across a span of gel, motivated by an across a span of gel, motivated by an electrical current. electrical current.

Activated electrodes at either end of the Activated electrodes at either end of the gel provide the driving force. gel provide the driving force.

A molecule's properties determine how A molecule's properties determine how rapidly an electric field can move the rapidly an electric field can move the molecule through a gelatinous medium.molecule through a gelatinous medium.

Gel ElectrophoresisGel Electrophoresis

Many important biological molecules such as Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionisable groups.nucleic acids, possess ionisable groups.

These molecules exist in solution as electrically These molecules exist in solution as electrically charged species either as cations (+) or anions charged species either as cations (+) or anions (-) at a given pH. (-) at a given pH.

The charged particles will migrate either to the The charged particles will migrate either to the cathode or to the anode depending on the cathode or to the anode depending on the nature of their net charge.nature of their net charge.


DNA, is mixed in a buffer solution and applied DNA, is mixed in a buffer solution and applied to a gel. to a gel.

The electrical current from one electrode The electrical current from one electrode repels the molecules while the other electrode repels the molecules while the other electrode simultaneously attracts the molecules. simultaneously attracts the molecules.

The frictional force of the gel material acts as a The frictional force of the gel material acts as a "molecular sieve," separating the molecules by "molecular sieve," separating the molecules by size. size.

During electrophoresis, macromolecules are During electrophoresis, macromolecules are forced to move through the pores when the forced to move through the pores when the electrical current is applied. electrical current is applied.


The rate of migration through the electric The rate of migration through the electric field field

depends on:depends on: The strength of the fieldThe strength of the field The size and shape of the moleculesThe size and shape of the molecules The ionic strength and temperature of the The ionic strength and temperature of the

buffer in which the molecules are moving. buffer in which the molecules are moving.

VisualizationVisualization

After staining, the separated After staining, the separated macromolecules in each lane can be seen macromolecules in each lane can be seen as a series of bands spread from one end as a series of bands spread from one end of the gel to the other.of the gel to the other.

The ladder is a mixture of fragments with The ladder is a mixture of fragments with known size to compare with the unknown known size to compare with the unknown fragments. fragments.

Gel after stainingGel after staining


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Southern blottingSouthern blotting

Southern blotting was named after Edward Southern blotting was named after Edward M. Southern who developed this procedure.M. Southern who developed this procedure.

To oversimplify, DNA molecules are To oversimplify, DNA molecules are transferred from an agarose onto a transferred from an agarose onto a membrane. membrane.

Southern blotting is designed to locate a Southern blotting is designed to locate a particular sequence of DNA within a particular sequence of DNA within a complex mixture. complex mixture.


For example, Southern Blotting could be used to For example, Southern Blotting could be used to locate a particular gene within an entire genome. locate a particular gene within an entire genome.

The amount of DNA needed for this technique is The amount of DNA needed for this technique is dependent on the size and specific activity of the dependent on the size and specific activity of the probe. Short probes tend to be more specific. probe. Short probes tend to be more specific.

Under optimal conditions, you can expect to Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are detect 0.1 pg of the DNA for which you are probing. probing.


Steps in Southern blotting:Steps in Southern blotting: Digest the DNA with an appropriate Digest the DNA with an appropriate

restriction enzyme restriction enzyme

Run the digest on an agaroseRun the digest on an agarose

Denature the DNA (usually while it is still on Denature the DNA (usually while it is still on the gel).For example, soak it in about 0.5M the gel).For example, soak it in about 0.5M NaOH. NaOH.

Only ssDNA can transfer.Only ssDNA can transfer.


fragments greater than 15 kb are hard fragments greater than 15 kb are hard to transfer to the blotting membrane. to transfer to the blotting membrane.

Depurination with HCl (about 0.2M HCl Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. cutting the DNA into smaller fragments.

However, that the procedure may also However, that the procedure may also be hampered by fragments that are too be hampered by fragments that are too small. small.

Southern blottingSouthern blotting Transfer the denatured DNA to the Transfer the denatured DNA to the

membrane. membrane.

Traditionally, a nitrocellulose membrane is Traditionally, a nitrocellulose membrane is used, although nylon membrane may be used, although nylon membrane may be used. Many scientists feel nylon is better used. Many scientists feel nylon is better since it binds more and is less fragile. since it binds more and is less fragile.

Transfer is usually done by capillary Transfer is usually done by capillary action, which takes several hours or using action, which takes several hours or using a vacuum blot apparatus which is faster). a vacuum blot apparatus which is faster).

Southern blottingSouthern blotting Capillary action transfer draws the buffer Capillary action transfer draws the buffer

up by capillary action through the gel into up by capillary action through the gel into the membrane, which will bind ssDNA.the membrane, which will bind ssDNA.

After you transfer your DNA to the After you transfer your DNA to the membrane, treat it with UV light. This membrane, treat it with UV light. This cross links (via covalent bonds) the DNA cross links (via covalent bonds) the DNA to the membrane. to the membrane.

(You can also bake nitrocellulose at about (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware 80C for a couple of hours, but be aware that it is very combustible.)that it is very combustible.)


Probe the membrane with labeled ssDNA. Probe the membrane with labeled ssDNA. This is also known as hybridization.This is also known as hybridization.

This process relies on the ssDNA This process relies on the ssDNA hybridizing (annealing) to the DNA on the hybridizing (annealing) to the DNA on the membrane due to the binding of membrane due to the binding of complementary strands.complementary strands.

Southern blotSouthern blot

Probe DetectionProbe Detection Visualize your labeled target sequence. Visualize your labeled target sequence.

Probing is often done with Probing is often done with 3232P labeled ATP, P labeled ATP, biotin/streptavidin or a bioluminescent probe. biotin/streptavidin or a bioluminescent probe.

If you used a radiolabeled If you used a radiolabeled 3232P probe, then you P probe, then you would visualize by autoradiograph. would visualize by autoradiograph.

Biotin/streptavidin detection is done by Biotin/streptavidin detection is done by colorimetric methods.colorimetric methods.

Bioluminescent visualization uses Bioluminescent visualization uses luminesence. luminesence.

Radioactive DetectionRadioactive Detection

Probe Detection using Probe Detection using Biotin/steptavidinBiotin/steptavidin

streptavidin is added which is an intermediary streptavidin is added which is an intermediary compound that will bind to the biotin on the compound that will bind to the biotin on the probe.probe.

Attached to the biotin is an enzyme such as Attached to the biotin is an enzyme such as alkaline phosphatase alkaline phosphatase

A chromogenic substrate for the enzyme is A chromogenic substrate for the enzyme is added eg. BCIP/NBT (for alkaline added eg. BCIP/NBT (for alkaline phosphatase), which produces a blue-purple phosphatase), which produces a blue-purple precipitate. precipitate.

Therefore, visualization does not require X-ray Therefore, visualization does not require X-ray film or other specific equipment.film or other specific equipment.

Biotin/Streptavidin detectionBiotin/Streptavidin detection

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Biotin/Streptavidin detectionBiotin/Streptavidin detection

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Northern BlottingNorthern Blotting

used for locating a sequence of RNA.used for locating a sequence of RNA.

It is also known as Northern hybridization It is also known as Northern hybridization or RNA hybridization. or RNA hybridization.

The procedure for and theory behind The procedure for and theory behind Northern blotting is almost identical to that Northern blotting is almost identical to that of Southern blotting, except you are of Southern blotting, except you are working with RNA instead of DNA.working with RNA instead of DNA.

Western BlottingWestern Blotting

Western blot analysis can detect oneWestern blot analysis can detect one protein in a mixture of any number of protein in a mixture of any number of proteins while giving you information proteins while giving you information about the size of the protein. about the size of the protein.

This method is, however, dependent on the This method is, however, dependent on the use of a high-quality antibody directed use of a high-quality antibody directed against a desired protein. against a desired protein.

Western BlottingWestern Blotting So you must be able to produce at least a So you must be able to produce at least a

small portion of the protein from a cloned small portion of the protein from a cloned DNA fragment to generate an antibody. DNA fragment to generate an antibody.

You will use this antibody as a probe to You will use this antibody as a probe to detect the protein of interest. detect the protein of interest.

Western blotting tells you how much Western blotting tells you how much protein has accumulated in cells.protein has accumulated in cells.

Steps in Western Steps in Western BlottingBlotting Separate the proteins using SDS-polyacrylamide Separate the proteins using SDS-polyacrylamide

gel electrophoresis (also known as SDS-PAGE. gel electrophoresis (also known as SDS-PAGE. This separates the proteins by size.This separates the proteins by size.

Place a nitrocellulose membrane on the gel and, Place a nitrocellulose membrane on the gel and,

using electrophoresis, drive the protein using electrophoresis, drive the protein (polypeptide) bands onto the nitrocellulose (polypeptide) bands onto the nitrocellulose membrane. membrane.

You want the negative charge to be on the side You want the negative charge to be on the side of the gel and the positive charge to be on the of the gel and the positive charge to be on the side of the nitrocellulose membrane to drive the side of the nitrocellulose membrane to drive the negatively charged proteins over to the negatively charged proteins over to the positively charged nitrocellulose membrane. positively charged nitrocellulose membrane.

Steps in Western Steps in Western BlottingBlotting

This gives you a nitrocellulose membrane This gives you a nitrocellulose membrane that is imprinted with the same protein that is imprinted with the same protein bands as the gel. bands as the gel.

Incubate the nitrocellulose membrane Incubate the nitrocellulose membrane with a primary antibody. with a primary antibody.

The primary antibody, which is the The primary antibody, which is the specific antibody mentioned above, sticks specific antibody mentioned above, sticks to your protein and forms an antibody-to your protein and forms an antibody-protein complex with the protein of protein complex with the protein of interest. interest.


Incubate the nitrocellulose membrane with a Incubate the nitrocellulose membrane with a secondary antibody. secondary antibody.

This antibody should be an antibody-enzyme This antibody should be an antibody-enzyme conjugate. conjugate.

The secondary antibody should be an antibody The secondary antibody should be an antibody against the primary antibody. against the primary antibody.

This means the secondary antibody will This means the secondary antibody will "stick" to the primary antibody, just like the "stick" to the primary antibody, just like the primary antibody "stuck" to the protein. primary antibody "stuck" to the protein.


The conjugated enzyme is there to allow you to The conjugated enzyme is there to allow you to visualize all of this. visualize all of this.

To actually see your enzyme in action, you'll need to To actually see your enzyme in action, you'll need to incubate it in a reaction mix that is specific for your incubate it in a reaction mix that is specific for your enzyme. enzyme.

You will see bands wherever there is a protein-You will see bands wherever there is a protein-primary antibody-secondary antibody-enzyme primary antibody-secondary antibody-enzyme complex, or, in other words, wherever your protein is. complex, or, in other words, wherever your protein is.

Put x-ray film on your membrane to detect a flash of Put x-ray film on your membrane to detect a flash of light, which is given off by the enzyme or observe the light, which is given off by the enzyme or observe the color change produced by the enzyme with the color change produced by the enzyme with the substrate.substrate.

Western blot procedureWestern blot procedurehttp://www.genscript.com/images/L00204-1.jpghttp://www.genscript.com/images/L00204-1.jpg

SDS Gel on left and Western blot SDS Gel on left and Western blot on righton right

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Steps in making a primary Steps in making a primary antibodyantibody

Run the purified protein on an SDS-PAGE Run the purified protein on an SDS-PAGE gel. gel.

Stain the gel with KCl. Stain the gel with KCl.

The KCl forms a precipitate with the SDS. The KCl forms a precipitate with the SDS.

Since the area with the protein has a low Since the area with the protein has a low concentration of SDS, the area with the concentration of SDS, the area with the protein will not show a precipitate. protein will not show a precipitate.

This will allow you to see the protein band This will allow you to see the protein band as a clear band against a milky white as a clear band against a milky white precipitate on the rest of the gelprecipitate on the rest of the gel. .


Carefully cut out the band, crush it and make Carefully cut out the band, crush it and make an emulsion with 1ml Freund's Complete an emulsion with 1ml Freund's Complete Adjuvant (which is an oily substance). Adjuvant (which is an oily substance).

The complete adjuvant contains bacteria (an The complete adjuvant contains bacteria (an immune stimulant) to increase the immune immune stimulant) to increase the immune response. response.

Inject this subscapularly into a rabbit. Inject this subscapularly into a rabbit.

This is your first inoculation. Only use the This is your first inoculation. Only use the complete adjuvant for the first inoculation. complete adjuvant for the first inoculation.


Rest the rabbit for one month, repeat the Rest the rabbit for one month, repeat the process using an incomplete adjuvant. You can process using an incomplete adjuvant. You can expect to see good antibody titers about 10 days expect to see good antibody titers about 10 days after the second booster. after the second booster.

Bleed the rabbit. Now you have rabbit antisera. Bleed the rabbit. Now you have rabbit antisera.

To get your primary antibody, dilute the rabbit To get your primary antibody, dilute the rabbit antisera in blotto (aka Carnation Nonfat Dry antisera in blotto (aka Carnation Nonfat Dry Instant Milk) and apply it to your nitrocellulose Instant Milk) and apply it to your nitrocellulose blot. Make sure you dilute 1:500 to 1:100 in blot. Make sure you dilute 1:500 to 1:100 in blotto.blotto.


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Steps in making secondary Steps in making secondary antibodyantibody

This is much easier than the procedure for the This is much easier than the procedure for the primary antibody. primary antibody.

Grab a catalogue and look for a goat-anti-rabbit Grab a catalogue and look for a goat-anti-rabbit antibody conjugated to horseradish peroxidase (HRP). antibody conjugated to horseradish peroxidase (HRP).

The goat-anti-rabbit is your secondary antibody (the The goat-anti-rabbit is your secondary antibody (the one that "sticks" to the primary antibody) and the HRP one that "sticks" to the primary antibody) and the HRP is the conjugated enzyme that will allow you to is the conjugated enzyme that will allow you to visualize your protein. visualize your protein.

DNA SequencingDNA Sequencing

determination of the precise sequence determination of the precise sequence of nucleotides in a sample of DNAof nucleotides in a sample of DNA

first devised in 1975, first devised in 1975, has become a powerful technique in has become a powerful technique in

molecular biologymolecular biology allows analysis of genes at the allows analysis of genes at the

nucleotide level. nucleotide level. has been applied to many areas of has been applied to many areas of

research. research.


Two methods:Two methods: Enzymatic sequencing/chain termination Enzymatic sequencing/chain termination

(or 'Sanger-Coulson-Sequencing')(or 'Sanger-Coulson-Sequencing') The sequence of a single-stranded DNA molecule The sequence of a single-stranded DNA molecule

is determined by enzymatic synthesis of is determined by enzymatic synthesis of complementary polynucleotide chains with these complementary polynucleotide chains with these chains terminating at specific nucleotide chains terminating at specific nucleotide positions.positions.

Chemical sequencing methodChemical sequencing method ('Maxam- ('Maxam-Gilbert-Sequencing’ Sequencing)Gilbert-Sequencing’ Sequencing)

The sequence of a double-stranded DNA molecule The sequence of a double-stranded DNA molecule is determined by treatment with chemicals that is determined by treatment with chemicals that cut the molecule at specific nucleotide positions. cut the molecule at specific nucleotide positions.


Both methods were equally popular to Both methods were equally popular to begin with begin with

However the chain termination However the chain termination procedure is currently preferred, procedure is currently preferred, particularly for genome sequencing. particularly for genome sequencing. because the chemicals used in the because the chemicals used in the

chemical degradation method are toxic chemical degradation method are toxic and therefore hazardous to the health of and therefore hazardous to the health of the researcherthe researcher

because it has been easier to automate because it has been easier to automate chain termination sequencing. chain termination sequencing.

Chain termination DNA Chain termination DNA sequencingsequencing

based on the principle that single-based on the principle that single-stranded DNA molecules that differ in stranded DNA molecules that differ in length by just a single nucleotide can be length by just a single nucleotide can be separated from one another by separated from one another by polyacrylamide gel electrophoresispolyacrylamide gel electrophoresis

The discovery of thermostable DNA The discovery of thermostable DNA polymerases, which led to the polymerases, which led to the development of PCR has also resulted in development of PCR has also resulted in new methodologies for chain termination new methodologies for chain termination sequencingsequencing


Thermal cycle sequencing has two Thermal cycle sequencing has two advantages over traditional chain advantages over traditional chain termination sequencing:termination sequencing: It uses double-stranded rather than It uses double-stranded rather than

single-stranded DNA as the starting single-stranded DNA as the starting material. material.

Very little template DNA is needed, so Very little template DNA is needed, so the DNA does not have to be cloned the DNA does not have to be cloned before being sequenced. before being sequenced.


Thermal cycle sequencing is carried out in a Thermal cycle sequencing is carried out in a similar way to PCR but :similar way to PCR but : just one primer is used just one primer is used each reaction mixture includes one of the ddNTPseach reaction mixture includes one of the ddNTPs Because there is only one primer, only one of the Because there is only one primer, only one of the

strands of the starting molecule is copiedstrands of the starting molecule is copied the product accumulates in a linear fashion, not the product accumulates in a linear fashion, not

exponentiallyexponentially The presence of the ddNTP in the reaction mixture The presence of the ddNTP in the reaction mixture

causes chain termination, causes chain termination, the family of resulting strands can be analyzed and the family of resulting strands can be analyzed and

the sequence read by polyacrylamide gel the sequence read by polyacrylamide gel electrophoresiselectrophoresis

Chain termination Chain termination sequencingsequencingThe strand synthesis reaction:The strand synthesis reaction:

is catalyzed by a DNA polymerase enzyme is catalyzed by a DNA polymerase enzyme requires the four dNTPs (dATP, dCTP, requires the four dNTPs (dATP, dCTP,

dGTP ,dTTP) dGTP ,dTTP) would normally continue until several thousand would normally continue until several thousand

nucleotides had been polymerized. nucleotides had been polymerized. However, this does not occur in a chain However, this does not occur in a chain

termination sequencing experiment because:termination sequencing experiment because: as well as the four dNTPs, a small amount of a as well as the four dNTPs, a small amount of a

dideoxynucleotide (e.g. ddATP) is added to the dideoxynucleotide (e.g. ddATP) is added to the reaction. reaction.

The polymerase enzyme does not discriminate The polymerase enzyme does not discriminate between dNTPs and ddNTPs, so the between dNTPs and ddNTPs, so the dideoxynucleotide can be incorporated into the dideoxynucleotide can be incorporated into the growing chain, but it then blocks further elongation growing chain, but it then blocks further elongation because it lacks the 3′-hydroxyl group needed to because it lacks the 3′-hydroxyl group needed to form a connection with the next nucleotide.form a connection with the next nucleotide.

The result is therefore a set of new chains, all of The result is therefore a set of new chains, all of different lengthsdifferent lengths

Chain termination Chain termination sequencingsequencing

Fluorolabeling has been important in Fluorolabeling has been important in the development of sequencing the development of sequencing methodology,methodology,

because the detection system for because the detection system for fluorolabels has opened the way to fluorolabels has opened the way to automated sequence reading. automated sequence reading.

The label is attached to the ddNTPs, The label is attached to the ddNTPs, with a different fluorolabel used for with a different fluorolabel used for each one. each one.

ChainChaintermination termination sequencingsequencing


Chains terminated with A are Chains terminated with A are therefore labeled with one therefore labeled with one fluorophore (pink), fluorophore (pink),

chains terminated with C are chains terminated with C are labeled with a second fluorophore labeled with a second fluorophore (blue), (blue),

chains terminated with G are chains terminated with G are labeled with another fluorophore labeled with another fluorophore (yellow)(yellow)

and chains terminated with T are and chains terminated with T are labeled with another fluorophore labeled with another fluorophore (green). (green).


Therefore, it is possible to :Therefore, it is possible to : carry out the four sequencing reactions - for A, C, G and T - carry out the four sequencing reactions - for A, C, G and T -

in a single tube in a single tube load all four families of molecules into just one lane of the load all four families of molecules into just one lane of the

polyacrylamide gel, because the fluorescent detector can polyacrylamide gel, because the fluorescent detector can discriminate between the different labels and hence discriminate between the different labels and hence determine if each band represents an A, C, G or T. determine if each band represents an A, C, G or T.

The sequence can be read directly as the bands pass The sequence can be read directly as the bands pass in front of the detector and either printed out in a in front of the detector and either printed out in a form readable by eye or sent straight to a computer form readable by eye or sent straight to a computer for storage. for storage.

When combined with robotic devices that prepare When combined with robotic devices that prepare the sequencing reactions and load the gel, the the sequencing reactions and load the gel, the fluorescent detection system provides a major fluorescent detection system provides a major increase in throughput and avoids errors that might increase in throughput and avoids errors that might arise when a sequence is read by eye and then arise when a sequence is read by eye and then entered manually into a computer. entered manually into a computer.

Automated DNA Automated DNA SequencerSequencer

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/F/FluorDideoxySeq.gif

Chain Chain TerminatiTermination on sequencinsequencingg

Sequence chromatogramSequence chromatogram

Animation of DNA Animation of DNA sequencingsequencing

http://www.dnalc.org/resources/http://www.dnalc.org/resources/animations/cycseq.htmlanimations/cycseq.html

ReferencesReferences

http://http://users.ugent.be/~avierstr/principles/users.ugent.be/~avierstr/principles/pcrsteps.gifpcrsteps.gif

http://http://www.flmnh.ufl.edu/cowries/PCR.gifwww.flmnh.ufl.edu/cowries/PCR.gif

UNIT 4 Techniques used in Molecular Biology. Objectives On completion of this unit students will be...

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Transcript of UNIT 4 Techniques used in Molecular Biology. Objectives On completion of this unit students will be...