Chapter 8 and 9: Tools By Which To Perform Experiments in Molecular Biology

I. Introduction

A. Tools of Molecular Biology: Introduction

1. As molecular biologists, we need techniques to study the following types of biological mechanisms

a. Gene expressionb. DNA replication

2. Experiments to study the chemical nature of the molecules of molecular biology better fall under the field of Biochemistry

3. The first question is where does one start with the study of molecular biology?

B. Tools of Molecular Biology: Introduction-Why Develop Experimental Tools

1. Molecular Biologists have developed a wide range of tools in order study the three basic molecules of Molecular Biology

a. DNAb. RNA c. Protein

2. Techniques in Molecular Biology have been developed for three reasons

a. Study basic gene functionb. Determine mechanisms of disease c. Disease diagnotics

3. In order to study basic gene function several classes of techniques had to be developed

a. Mechanisms to clone genesb. Mechanisms by which to control gene expression

4. They needed tools to determine the root cause of the disorders they wanted to study

a. If following a chromosomal disorder-they needed tools to see whether there were too many or too few chromosomes (or chromosome parts)

b. For single gene disorders, they needed tools to be able to find the mutation in the gene, and to determine how that mutation may affect the expression of the gene of interest

5. Some of the same tools that would be used to determine the root cause can be used in diagnostics

a. Can be used to confirm or determine whether one has disease causing alleles

b. Can be used for preventative purposes

6. Some of the tools can also be used in forensics

7. Supplemental Figure: Tools of Molecular Biology: Introduction-Why Develop Experimental Tools

C. Tools of Molecular Biology: Introduction-Where To Start

1. In Molecular Biology, one starts with the study of a gene or genes of interest

2. The difficulty with this is that we need to get the genes isolated from the genome and placed into a system which is more amenable to manipulation

3. The process of isolating genes from a genome and then placing them in a system that is more amenable to manipulation is known as molecular cloning

4. Obtaining the gene from the genomic DNA can happen by two general approaches

a. Amplifying (creating large numbers of copies) the gene using the genomic DNA as a template (most common)b. Directly cutting the gene out of the genomic DNA

5. Once the DNA encoding the gene is isolated it is then placed into another DNA molecule that serves as a vector

a. The result is a recombinant DNA molecule

b. By definition, a recombinant DNA molecule is a single DNA molecule in which 2 or more portions of the molecule come from different origins

6. Tools of Molecular Biology: Introduction-Historical Perspectives Into Molecular Cloning

7. In the early 1960s, before the advent of molecular cloning, genetic studies at the molecular level relied on indirect or fortuitous discoveries

a. The ability of bacteriophages to incorporate bacterial genes into their genomes

b. Phage phi 80 was observed with the lac operator from E. coli integrated into its genome

8. The development of recombinant DNA technologies began with a seminal discovery in 1962 in Allen Campbell’s lab

a. The bacteriophage genome, which is linear actually λcircularizes when inserted into a host bacterial cell

b. Further analysis bacteriophage had short regions of λsingle stranded sequences which were complementary to each other

c. This complementary single-stranded sequence on each end of the the bacteriophage DNA can base pair and thus λare called cohesive sites (cos sites)

d. The binding of the cohesive sites allows for the bacteriophage DNA to circularizeλ

9. The idea of joining DNA segments by cohesive sites became crucial to the development of recombinant DNA technology as it shed light on a method by which to combine two segments of DNA from different sources

10. Today, we use restriction enzymes, which were initially discovered by Werner Arber, to create compatible ends for creating recombinant DNA molecules

11. Through many experimental observations between 1972-1975 by research groups studying the bacteriophage , the λprocess of molecular cloning was refined

II. Molecular Cloning

A. Molecular Cloning: Obstacles Molecular Geneticists Face When Studying DNA

1. Molecular genetics, when studying DNA face the obstacle of having very little material to start with

2. Starting material is the genomic DNA-this is all the DNA the makes up the individuals genome (contains all the persons genes)

3. Each person has only two copies of each chromosome when studying chromosomal disorders and so we need methods to visualize chromosomes at high resolution

4. For each gene, each individual has only two copies, and so we need methods to be able to study such a small number of copies when following a single-gene disorder

5. Also, we need techniques to be able to specifically follow a single chromosome pair or a single gene from all others

B. Molecular Cloning: Basic Tools For Molecular Molecular Cloning

1. Given the size of any genome, it is quite hard to work with it when trying to study a single gene

2. There are two tools that allow us to study a single gene at a time

3. The first tool are restriction enzymes

4. The second tool are vectors (pieces of DNA in which you can place your gene of interest)

C. Molecular Cloning: General Techniques Used To Study DNA: Restriction Enzymes

1. An important advance in the study of molecular genetics was the discovery of restriction enzymes in the 1970s

a. The enzymes were discovered in bacteriab. They function to cut DNA at SPECIFIC Sequences

2. Today, we know of more than 3500 restricition enzymes

3. There are three classes of restriction enzymes

a. Type Ib. Type IIc. Type III

4. Type I restriction enzymes cut DNA at sites that are far from their recognition sequence

5. Type II restriction enzymes recognize specific DNA sequences and cut within (or near) those sequences

6. Type III restriction enzymes need to recognize two different sequences in opposite orientations and will cut outside these sequences

7. Of the 3500 restriction enzymes identified, only a small percentage are commercially available for use in laboratory experiments

a. There are 240 type II restriction enzymes, which compose a great percentage of those available

b. A small percentage of those commercially available are type I restriction enzymes

8. Each type II restriction endonuclease is composed of two identical peptide subunits that join together to form a homodimer

9. Each restriction endonuclease is named for the organism in which they were discovered, using a system of letters and numbers

10. HindIII was discovered from Haemophilus influenza (strain D)

a. The Hin comes from the first letter of the genus name and the first two letters of the species nameb. The d comes from the strain typec. The III comes from the fact that it is the third restriction enzyme isolated from this organism

11. EcoRI was isolated from E. coli strain R and is the first enzyme isolated from this organism

12. Most of the type II restriction enzymes recognize sequence of either 4 or 6 base pairs, however some recognize sequences of 8 or 10 base pairs in length

13. The most commonly used restriction enzymes recognize sequences 6 base pairs

14. The sequences the restriction enzymes recognize are palindromes; which means the sequences in the recognition site when read in the 5’ 3’ direction are the same for both strands of the double stranded DNA molecule

a. EcoRI is a restriction enzyme that recognizes the sequence 5’ GAATTC 3’

b. The complementary strand also has the sequence 5’ GAATTC 3’

15. To find out how frequently a type two restriction enzyme we use the formula 4n

a. The 4 stand for the fact that at a single position with in the DNA, there is the possibility of 1 of 4 possible bases being presentb. The n stands for the number base pairs present in the restriction sequence

16. For instance, The human genome has 3 billion base pairs how often should any one restriction enzyme cut the DNA within the human genome?

17. One can determine, on average, how often a restriction enzyme should cut

a. For EcoRI, it cuts every six basesb. It should cut on average every 46 base pairs (or every 4096 base pairs) c. 4 is equal to the number of possible bases at a position in the DNA and the 6 is for the bases in the recognition sited. Therefore, EcoRI should cut the human genomic DNA approximately 730,000 times (3,000,000,000/4096)

D. Molecular Cloning: Cutting DNA With Restriction Enzymes

1. When digesting the DNA with restriction enzymes, one can consistently predict the possible products that are going to result from the restriction enzymes

a. The restriction enzymes recognize specific sequences b. The restriction enzyme cuts the DNA at the site in a consistent manner (the same way every time)

2. Again, the EcoRI restriction enzyme recognizes the site 5’ GAATTC 3’ (The site is double stranded)

3. When the restriction enzyme cuts, it cuts at this site by cleaving the phosphodiester backbone

4. Within the sequence, the EcoRI enzyme will cut the phosphodiester backbone at a specific place

5. The enzyme cuts the backbone between the G and the A on both strands leaving a 5’ overhang

a. The 5’ overhang is single stranded sequence that is left on the products (the DNA molecules left after using the restriction enzyme) after cutting the DNA with the EcoRI

b. Some restriction enzymes are blunt cutters, leaving no overhangs

c. Some enzymes leave 3’ overhangs

6. If one were to have a piece of DNA with two EcoRI sites 1000 base pairs apart, after cutting with EcoRI, you will have a 1000 base pair fragment produced after cutting with the EcoRI enzyme

7. Supplemental Figure: Cutting a Gene Using Restriction Enzymes

E. Molecular Cloning: Restriction Site Geometry

1. Each individual restriction enzyme will recognize its own specific sequence, and will cut that sequence at a specific place

2. HindIII recognizes the sequence 5’-P-AAGCTT-OH-3’

a. HindIII cuts between the two A residuesb. HindIII leaves 5’ overhangs

3. SmaI recognizes the sequence 5’-P-CCCGGG-OH-3’

a. SmaI cuts between the C and the Gb. SmaI leaves blunt ends

4. SacI recognizes the sequence 5’-P-CTCGAG-OH-3’

a. SacI cuts between the A and the Gb. SacI leaves 3’ overhangs

5. Each restriction site has an axis of symmetry

6. It is easy to predict what type of overhangs will be present depending where the restriction enzyme cuts the restriction recognition sequence with respect to the axis of symmetry

a. If the restriction enzyme cuts 5’ to the axis of symmetry, then 5’overhangs will result

b. If the restriction enzyme cuts at the axis of symmetry, then blunt ends will result

c. If the restriction enzyme cuts 3’ to the axis of symmetry, then 3’ overhangs will result

F. Molecular Cloning: Materials Needed For the Cloning To Take Place

1. In molecular cloning procedures, two different molecules are needed

a. Insertb.Vector

2. Insert will be the gene that has been isolated from the genomic DNA (as we previously saw)

3. The vector will be the specific DNA molecule in which the gene will be placed

G. Molecular Cloning: Information About Vectors

1. A vector is a DNA molecule that can replicate autonomously in a host such as bacterial cells

2. These replicating vectors can often achieve a high number of copies per cell, and can be easily isolated from the cells for further analysis

3. Several different types of vectors can be used

a. Plasmidsb. Cosmidsc. BACs (Bacterial Artificial Chromosomes)d. YACs (Yeast Artificial Chromosomes)

4. Plasmids are usually less than 10 kb and are generally used for subcloning purposes

5. Cosmids are 35-45 kb and are used for genomic library construction

6. BACs are 75-300 kb and are used for analysis of large genomes (cloning of large genome fragments)

7. YACs are 100-1000 kb and are also used in the analysis of large genomes (cloning of large genome fragments)

H. Molecular Cloning: Using Plasmids As A Vector

1. The most common form of vector that is used for cloning purposes is a plasmid

2. Each type of plasmid is specific for its host

a. Specific plasmids for bacteria, which are in the size range of 3-5 kb

b. Specific plasmids for yeast, which are in the size range of 5-10 kb

3. Plasmids are circular double stranded molecules that exist separately from the host genome and replicate independently from the host genome

4. Plasmids are derived from naturally occurring DNA molecules that were first discovered in bacteria due to the fact that they carried antibiotic resistance genes

5. Plasmids can be passed from one bacterium to another

6. Each plasmid contains three basic properties

a. Origin of replicationb. A gene that encodes for a selectable markerc. A region called the multiple cloning site which contains many restriction sites so you can put your gene of interest into the plasmid

J. Molecular Cloning: Inserting the Gene Into The Vector

1. The purpose of molecular cloning is to isolate a particular gene or other DNA sequence from the genome and place it in a vector

2. The gene is cut from the genomic DNA using either one or two different restriction enzymes

a. If one enzyme is used then both ends of the DNA fragment carrying the gene will have the same ends

b. If two enzymes are used, then the ends of the DNA fragment will have different ends

3. In order to place this fragment into the plasmid, then the plasmid must also be cut with the same enzymes

a. Create compatible ends between the insert and the vector

b. It is necessary to check whether the vector has the appropriate sites in its MCS before cutting the gene from the genomic DNA

4. The restriction enzyme that is used to cut out your sequence and cut the plasmid is usually one that leaves an overhang

a. 5’ overhangb. 3’ overhang

5. These overhangs allow for easier ligation (joining of your gene into the plasmid)

6. To ligate your gene and the plasmid together, the enzyme T4 DNA ligase is used-this allows joining of your plasmid and gene of interest (insert)

7. The goal of T4 DNA ligase is to form phosphodiester bonds between 5’ PO4 and 3’ OH groups

a. During a ligation reaction, 4 phosphodiester bonds need to be formed

b. T4 DNA ligase catalyzes the formation of phosphodiester bonds between ends that contain overhangs more efficiently

c. T4 DNA ligase catalyzes the formation of phosphodiester bonds between blunt ends less efficiently

8. After ligation, the product is a recombinant molecule containing the following

7. If only one restriction enzyme is used to digest the insert and vector, then there are two possible orientations of your gene of interest in the recombinant DNA molecule

8. If two different restriction enzymes are used to digest both insert and vector, then there will be only one possible orientation for your gene in the recombinant DNA molecule

K. Supplemental Figure: Molecular Cloning: Overview of the Entire Process

L. Molecular Cloning: Obtaining Enough Gene Sequence To Clone

1. If one wants to clone a specific gene, it may not be efficient to just cut the specific gene out of the genomic DNA using restriction enzymes

2. This is because there are generally not restriction enzyme sites located exactly on the ends of your gene of interest

3. In a tube of genomic DNA, there are also very few copies of your gene of interest compared to the rest of the genome sequences

4. Therefore, we must find a way to amplify your gene of interest before molecular cloning It into a vector

M. Molecular Cloning: Obtaining Enough Gene Sequence To Clone (PCR)

1. To amplify (make copies) of your gene of interest, one can use the polymerase chain reaction (PCR)

2. PCR was developed by Kary Mullis in the early 1980s

3. In a PCR reaction, we place several components

a. Genomic DNAb. Nucleotides (the building blocks of DNA)c. Taq DNA polymerase (enzyme that carries out the reaction and is thermostable in that it can withstand high temperaturesd. A buffer that is optimized for Taq activitye. Primers

4. The primers small DNA molecules of about 20 nucleotides

5. They are specific for the gene of interest because they are designed to be complementary to the ends of your gene of interest

6. There are two primers in the reaction, the forward primer and the reverse primer

7. The forward primer has complementary sequence to the DNA strand that goes 5’ 3’ in the right to left direction, and it binds on the 3’ end to its complementary sequence

8. The reverse primer has complementary sequence to the DNA strand that goes 5’ 3’ in the left the right direction on the 3’ end

9. Once the reaction is set-up, we will then run the reaction in a DNA thermocycler, and instrument that can precisely and quickly change the temperature at which the reaction is being run at

10. To start the reaction, we bring the temperature up to 95 C

11. This acts to denature the DNA-i.e. the heat will break the hydrogen bonds between the two strands of DNA to make them single stranded

12. The single stranded DNA molecules will be the templates for the reaction

13. Next, the temperature is cooled to about 55 C – 60 C to allow the primers to bind to their complementary sequence (30S)

14. Once the primers have bound their complementary sequence, we move the reaction up to 72 C

15. 72 C is the optimal temperature for Taq DNA polymerase to work

16. The Taq DNA polymerase creates new DNA sequence by binding onto the 3’ ends of the primers, and extending the DNA sequence

17. The taq DNA polymerase extends the DNA sequence in the 5’ 3’ direction creating sequence complementary to the template

18. If you were to start with one molecule of DNA, after one round of PCR, you would have 2

19. We often repeat these steps where we change temperature about 30 times

20. In the end we have 230 copies of our gene of interest

21. These copies can then be subjected to restriction digestion and can be cloned into a vector to make a recombinant DNA molecule

N. Molecular Cloning: Transforming The Ligation Reaction

1. In a ligation reaction, only a very small amount of recombinant plasmid is produced

a. Usually only nanogram or picogram quantities of recombinant plasmid

b. Normally in the lab people work with microgram quantities of DNA

2. Also, we want to separate each newly made recombinant plasmid from one another

3. In order to separate the recombinant plasmids and and amplify the amount of each a transformation reaction is performed

4. In a transformation reaction, we are inducing E. coli bacteria to uptake our recombinant plasmid

5. In order to select the bacteria containing the plasmid are selected using antibiotic

a. The term transformation comes from the fact that the bacteria are uptaking foreign DNA which makes them genetically different from the original strain and provides antibiotic resistance

b. In most cases this is ampicillin

c. In most cases the plasmid contains the amp resistance gene

6. Once transformed the bacteria will replicated the plasmid and as well, will pass the plasmid onto their daughter cells which will also replicate the plasmid

7. The bacterial strain that is used for transformation of ligations is the E. coli DH5α

8. There are two methods by which to transform cells

a. Electroporationb. Chemical transformation using calcium

9. Most transformations are accomplished by using calcium competent cells

O. Molecular Cloning: Transforming The Ligation Reaction Using Calcium Competent Cells

1. The transformation using calcium competent cells follows the following steps:

2. Mix ligation reaction and competent cells together and incubate on ice to allow the DNA to bind to the surface of the E. coli cell

3. Competent cell/ligation reaction mixture are heat shocked at 40 C for 1-1.5 minutes to allow for uptake of the recombinant DNA

4. Cells are then mixed with LB broth at are subjected to shaking at 37 C for 30 mins.-1 hr. to allow the cells to recover from the heat shock

5. Transformation mixture is plated on LB-AMP media and incubated at 37 C to select for cells containing intact plasmid (please note that cut plasmid that has not ligated back together will not successfully transform)

III. Searching a Library of Clones

A. Searching A Library of Clones: Introduction

1. Depending upon what we want to study will determine what we will clone

2. If one wants to study, the whole genome, one can make a genomic library

a. To make a library, one digests the genomic DNA with a restriction enzyme

b. The fragments created by the restriction enzyme digest will be ligated into the vector

c. This creates the library of clones that you can search for your gene of interest, or that you can used for genome sequencing

3. If you want to find a recombinant plasmid (clone) containing your gene of interest, you must have a way of searching for the recombinant plasmid containing your gene of interest

4. The experimental technique that allows us to find a recombinant plasmid containing your gene of interest is the Southern Blot

B. Searching a Library of Clones: The Southern Blot

1. Southern blotting was developed in the 1975 by Edward Southern

2. The goal of the southern blot technique allows one to find an examine specific DNA fragments amongst a large number of DNA fragments

a. These fragments are usually derived from the genomic DNA from the appropriate source

b. The DNA fragments are produced by restriction digests

3. From this, the recombinant plasmid containing your gene of interest can be specifically found

4. Once one obtains the genomic DNA of choice, the first thing is to digest it using restriction enzyme(s)

5. The next step is to then run the DNA sample on an agarose gel

a. The gel is a matrix that allows the pieces to be separated by size

b. The smaller the size, the faster and further the fragment will run on the gel

c. The large the size, the slower and shorter the DNA fragment will run

d. Given that there are many fragments created by digestion of genomic DNA of all sizes, one will not be able to pick out one individual fragment after staining the gel with ethidium bromide-instead one will see a smear on the gel

6. At this point the DNA is not in a state where it can be analyzed

7. To be analyzed, it must be first denatured such that it become single stranded (usually by using 0.4 N NaOH)

8. Once the DNA has been denatured to become single stranded, they are transferred to a nylon membrane by capillary action

a. The gel is placed on top of a wet bed of Watman Paper b. To transfer the nylon membrane is placed directly on top of the gelc. On top of the membrane, a stack of paper towels and a weight is placedd. In this setup, the liquid in the Watman moves up from the Watman to through the gel to the nylon membrane carrying the single stranded DNA with it

9. Once the DNA is transferred to the membrane, it is then cross-linked to the membrane

10. In the Southern Blot, we want to indentify a specific DNA fragment(s) from the million fragments produced by the restriction digest

11. To identify the specific fragment(s) the blot must be probed with a single stranded DNA probe that is complementary to the gene you are interested in studying

a. This probe doesn’t have to cover the full length of the gene

b. The probe is labeled with radioactivity (32P) such that you can use X-ray film or a phosphorimager to visualize your results

12. Binding of the probe to complementary sequence (hybridization) will mark the location of the DNA fragment you are interested in on the blot

13. Where your fragment is you will see a band on the developed X-ray film, or on your phosphorimager scan

14. Supplemental Figure Box 8.7: Searching a Library of Clones: The Southern Blot

IV. DNA Sequencing

A. DNA Sequence Analysis: Introduction

1. In Human Molecular Genetics, DNA sequencing is used for two main purposes

a. To determine the sequence of a gene or genome of interestb. If a patient has a disorder, to find the mutation in the appropriate gene

2.The most widely used approach for DNA sequencing is the one developed by Fred Sanger and Walter Gilbert

3They initially published their work in 1975

4. For developing DNA sequencing, Sanger and Gilbert won the Nobel Prize in Medicine in 1980

5. DNA sequencing, on a technical level, builds upon the principles of PCR

B. DNA Sequence Analysis: General Methodology

1. The key to DNA sequencing is a special nucleotide; the dideoxynucleotide (ddNTP)

2. The dideoxynucleotide is similar to a deoxynucleotide except for one main difference

3. At the 3’ Carbon, there is a hydrogen instead of a hydroxyl group

4. If this dideoxynucleotide is added during an elongation step in a PCR reaction, elongation of the chain stops

5. Elongation stops because there is no OH group at the 3’ carbon to create a phosphodiester bond with

6. In Sanger sequencing, much like a PCR reaction, the fragment or clone to be sequenced also serves as a template

7. For instance, if you want to sequence a certain gene, the DNA encoding that gene is your template

8. In this reaction, we will add a primer

a, This is different from PCR in which one uses two primersb. The only primer that is used is the forward primer because we want to sequence the coding strand

9. As well, a DNA polymerase is added, a buffer is added, as well as normal deoxynucleotides

10. In addition the dideoxy-nucleotides are also added to the reaction (ddATP, ddTTP, ddCTP, ddGTP)

11. Each of these dideoxy-nucleotides are labeled with a fluorescent dye of a different color

12. To sequence your gene, the DNA is first denatured, then your primer is then annealed to your template (similar to PCR)

13. Then, from the DNA polymerase is able to bind the template and synthesize a new DNA strand using the 3’ end of the primer

14. For each position in the growing DNA strand, either a deoxynucleotide or a dideoxynucleotide can be added that is complementary to the template

a. If a deoxynucleotide is added, then the DNA strand can be further elongated

b. If a dideoxynucleotide is added, then the DNA strand can no longer be elongated

15. What happens is that one gets a whole set of newly synthesized single stranded DNA molecules of different sizes (remember they are single stranded because there is no reverse primer as there is in PCR)

16. Theoretically, each of these fragments will be different in size by one nucleotide (if you had a gene that is 1000 nucleotides long, you would end up with roughly 1000 fragments)

17. Once the reaction is completed, the DNA fragments are run on a gel

18. The smaller fragments run faster and further and the larger fragments slower and shorter

19. Once the gel is run, it can be analyzed by a machine that can read the flourescence from the dideoy-nucleotide that has been incorporated into each fragment

20. You can find the sequence of your gene by reading the gel from bottom to top based on the dideoxy-nucleotide that was added

V. Techniques to Study Gene Expression

A. Techniques To Study Gene Expression: Introduction

1. There are two general methods by which to study gene expression

a. Study gene expression by following the appropriate molecules produced endogenouslyb. Study gene expression by using recombinant molecules

2. When molecular biologists study gene expression, they use both general methods to get a full picture of what is happening

3. Using our clones (recombinant plasmids containing our genes of interest) allows us to more flexibility in performing our experiments

a. Selectively turn expression of our gene on and off at our choosing

b. Selectively make specific mutations in our gene

4. Although you can more easily manipulate the system when using recombinant plasmids containing your gene of interest, you can create artifactual data

5. The process of gene expression occurs in three steps

6. First, the gene, which is encoded in DNA is transcribed to produce an mRNA

7. Second, the mRNA is shuttled out of the nucleus and translated to produce a protein

8. Third, the protein is the molecule that carries out the function of the gene (and this protein may be modified before it is fully functional)

a. Phosphorylationb. Cleavage

9. The ultimate goal of gene expression is to get to a functional protein

B. Techniques To Study Gene Expression: An Introduction To Following Endogenous Molecules

1. When following gene expression in vivo, it can be very effective to study endogenous molecules

2. Since gene expression follows this central dogma: DNA mRNA Protein, there are two endogenous molecules one can follow

a. mRNAb. Protein

3. Following protein gives the most direct readout on gene expression as this is the molecule that carries out the function of the gene

a. mRNA may be present, but can be translationally controlled

b. Following mRNA can be more cost-effective if one can work without degrading the mRNA

C. Techniques To Study Gene Expression: Introduction To Following mRNA By Northern Blot

1. The goal of the Northern Blot is to study the expression of a particular mRNA amongst all of the mRNA present in the cell

a. The strategy is most commonly used to determine mRNA levels

b. mRNA levels are oftentimes proportional to the level at which the gene is expressed because the mRNA is translated to make protein

c. Less commonly, the northern blot technique is used to determine mRNA size

2. The methodology for doing a Northern Blot is quite similar to that of a Southern Blot except we will again be following RNA

3. Since the technique is based off of the Southern blot (developed by Edward Southern-1975), it became known as the northern blot

4. Method is easy and produces results that are easily reproducible-most common method used to study cellular mRNA levels

D. Techniques To Study Gene Expression: Northern Blot Analysis-Procedural Overview

1. First RNA isolations must be performed from the cell line/strain of choice

a. Allows us to have a sample of cellular RNA (all types)

b. The result is that we have purified RNA (only ~2% of RNA is mRNA in yeast, ~10% in other species)

2. Once the total RNA is isolated, then we run the RNA sample on the gel

a. If we stain the gel with ethidium bromide, we will see the ribosomal RNA as bands on the gel

b. The mRNA, since the levels are so low, will not be seen by staining

c. The gel contains formaldehyde, which is a denaturant

3. The RNA is transferred to a nylon membrane and then crosslinked to the membrane

a. Transfer occurs in a similar manner to a Southern Blot

b. Transfer occurs by capillary action

4. Lastly, the blot is probed using a probe complementary to the mRNA of interest

E. Techniques to Study Gene Expression: Transferring the RNA To The Nylon Membrane (agarose gel)

1. The Northern Transfer is performed in a similar manner to the Southern transfer

2. Create a transfer apparatus by using a tray and making a paper wick with Whatman Paper

3. In the tray, place 10X SSC-this is the solution that will be used for the transfer

a. SSC stands for salt (NaCl), Sodium Citrate

b. This is just a salt buffer and does not need to function as a denaturant since the formaldehyde in the gel performed the process

4. Place gel on bed of wet whatman paper

5. Place nylon membrane on top of the gel

6. Place three whatman papers on top of the gel

7. Place a stack of paper towels on top of the membrane

8. Place a weight on top of the paper towels

9. 10X SSC will move out of the tray and up through wick, gel, membrane and paper towels by capillary action-RNA will move out of the gel and be transferred to membrane

10. The RNA is then usually crosslinked to the gel by using UV light

F. Techniques To Study Gene Expression: Probing the Northern Blot

1. In order to probe the blots, two types of probes can be used

a. RNA probes with complementary sequence to the mRNA of interestb. Oligo probes with complementary sequence to the mRNA of interest

2. RNA probes are made by in vitro transcription in the presence of 32P radio-labeled nucleotide (s) – usually use the nucleotide that is most frequently found in the mRNA to be probed –good for studying less abundant transcripts

3. Oligo probes are short (~20-30 nt) and composed of DNA

a. To add a radiolabel to the oligo use 32P-labeled dATP and T4 DNA Kinaseb. Easy to make and good for studying abundant transcripts

4. Alternatively an HRP-conjugated nucleotide can be used which will allow for detection by colormetric assay

5. If RNA is present in the RNA sample(s) then it will appear as a band

G. Techniques to Study Gene Expression: Introduction to Western Blotting

1. The other commonly used method to study gene expression is the western blot

a. A western blot is used to probe for proteins

b. The southern blot is used to probe for DNA and a northern blot is used to probe for RNA

2. Because the western is used to study protein levels, it is a more direct way to study gene expression

3. The general paradigm is quite similar to Northern/Southern lab, however the methodology is quite different

a. Similar the other two procedures, proteins are run on a gel, then transferred to a membrane and probed

b. The gel that is run is an SDS-polyacrylamide gel

c. The blot is probed with protein specific antibodies

H. Techniques to Study Gene Expression: Creating Protein Specific Antibodies For Western Blots

1. For the protein you are interested in study, obtain purified protein

2. Inject the purified protein into a rabbit

3. Rabbit’s immune system will recognize that the protein is foreign and will make antibodies against the protein

4. Take blood samples from the rabbit and then purify antibodies from the blood samples

5. Antibodies produced are considered polyclonal antibodies because each protein that is injected a mixture of antibodies will made that recognize different epitopes within the protein

J. Techniques to Study Gene Expression: Western Blotting Procedures

1. First, protein must be extracted from the cell line of choice to make an extract

2. Next, a sample of extract is then mixed with SDS-sample buffer and boiled

a. The sample buffer contains dyeb. The sample buffer contains SDS, which is a detergentc. The SDS acts to denature the proteind. The SDS also coats the protein, giving it a net negative chargee. The boiling step also aids in the denaturation of the protein

3. Once the proteins are denatured, the sample above is run on an SDS-polyacrylamide gel

a. The gel contains SDS which again acts as a denaturant, which allows proteins to run true to sizeb. Larger proteins run shorter on the gelc. Smaller proteins run further on the geld. Electrical current is used to run the proteins on the gel-since the proteins have a net negative charge, they run towards the positive electrode

4. Once the gel is run, the proteins are transfer horizontally to a nitrocellulose membrane

a. Proteins are transferred to nitrocellulose, whereas nucleic acids are transferred to a nylon membraneb. Proteins are transferred by using electrical currentc. The transfer is done horizontally towards the anode, as the proteins are coated with the negatively charged SDS

5. Supplemental Figure: Techniques to Study Gene Expression: Western Blotting Procedures

6. In order to visualize your proteins on the blot, the blot must be probed

a. For Western Blots, antibodies are usedb. For Northern and Southern Blots, nucleic acid probes are used

7. In order to get an efficient probing of the blot, two antibodies are used

a. The primary antibody binds the protein of interestb. The secondary antibody binds the constant region of the primary antibodyc. The constant region of an antibody is held in common for all antibodies produced by organisms of the same speciesd. The secondary antibody has either Alkaline phosphatase or Horseradish peroxidase conjugated to it

8. Once the antibodies are bound, the protein of interest can be visualized by two methods

a. Using a chemiluminescent substrate or a colorimetric substrateb. The conjugated enzyme will oxidize the substrate and if it is the chemiluminescent substrate, light will be emittedc. The conjugated enzyme will oxidize the substrate and if it is the colorimetric substrate, then a purple product is left behind

9. In either case, the protein of interest will appear as a band on the blot

10. Supplemental Figure 2: Techniques to Study Gene Expression: Western Blotting Procedures