Molecular Biology Fifth Edition Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 5 Molecular Tools for Studying Genes and Gene Activity Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

5.1 Molecular Separations • Often mixtures of proteins or nucleic acids are generated during the course of molecular biological procedures A protein may need to be purified from a crude cellular extract A particular nucleic acid molecule made in a reaction needs to be purified Gel electrophoresis is used to separate different species of: Nucleic acid Protein

DNA Gel Electrophoresis Melted agarose is poured into a form equipped with removable comb Comb “teeth” form slots in the solidified agarose DNA samples are placed in the slots An electric current is run through the gel at a neutral pH to allow the sample to travel through the gel matrix

DNA Separation by Agarose Gel Electrophoresis DNA is negatively charged due to the phosphates in its backbone and moves toward the positive pole Small DNA pieces have little frictional drag so they move rapidly Large DNAs have more frictional drag so their mobility is slower Distributes DNA according to size Largest near the top Smallest near the bottom DNA is stained with fluorescent dye that intercalates between the bases

DNA Size Estimation Mobility of fragments are plotted v. log of molecular weight (or number of base pairs) Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation upon comparison Same principles apply to RNA separation

Electrophoresis of Large DNA Special techniques are required for DNA fragments larger than about 1 kilobases Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction Technique is called pulsed-field gel electrophoresis (PFGE)

Protein Gel Electrophoresis Separation of proteins is done using polyacrylamide gel electrophoresis (PAGE) Treat proteins to denature subunits with detergent such as sodium dodecyl sulfate (SDS) SDS coats polypeptides with negative charges so all move to anode Masks natural charges of protein subunits so all move relative to mass not charge As with DNA smaller proteins move faster toward the anode

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

Two-Dimensional Gel Electrophoresis While SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is needed Two-dimensional gel electrophoresis: Nondenaturing gel electrophoresis (no SDS) uses 2 consecutive gels each in a different dimension Sequential gels with distinct pH separation and polyacrylamide gel concentration

A Simple 2-D Method Samples are run in 2 gels First dimension separates using one concentration of polyacrylamide at one pH Second dimension uses different concentration of polyacrylamide and pH Proteins move differently at different pH values without SDS and at different acrylamide concentrations

A More Powerful Two-Dimensional Gel Electrophoresis Technique A two process method: Isoelectric focusing gel: mixture of proteins electrophoresed through gel in a narrow tube containing a pH gradient Negatively charged protein moves to its isoelectric point at which it is no longer charged Tube gel is removed and used as the sample in the second process

A More Powerful Two-Dimensional Gel Electrophoresis Technique continued Standard SDS-PAGE: Tube gel is removed and used as the sample at the top of a standard polyacrylamide gel Proteins partially resolved by isoelectric focusing are further resolved according to size When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible

Ion-Exchange Chromatography Chromatography originally referred to the pattern seen after separating colored substances on paper Ion-exchange chromatography uses a resin to separate substances by charge This is especially useful for proteins Resin is placed in a column and the sample is loaded onto the column material

Separation by Ion-Exchange Chromatography Once the sample is loaded buffer is passed over the resin + sample As ionic strength of elution buffer increases, samples of solution flowing through the column are collected Samples are tested for the presence of the protein of interest

Gel Filtration Chromatography Protein size is a valuable property that can be used as a basis of physical separation Gel filtration uses columns filled with porous resins that let in smaller substances and exclude larger substances As a result larger substances travel faster through the column

Affinity Chromatography The resin contains a substance to which the molecule of interest has a strong and specific affinity The molecule binds to a column resin coupled to the affinity reagent Molecule of interest is retained Most other molecules flow through without binding Last, the molecule of interest is eluted from the column using a specific solution that disrupts their specific binding

Summary High-resolution separation of proteins can be achieved by two-dimensional gel electrophoresis Ion-exchange chromatography can be used to separate substances according to their sizes Gel filtration chromatography uses columns filled with porous resins that let in smaller substances but exclude larger ones Affinity chromatography is a powerful purification technique that exploits an affinity reagent with strong and specific affinity for a molecule of interest

5.2 Labeled Tracers For many years “labeled” has been synonymous with “radioactive” Radioactive tracers allow vanishingly small quantities of substances to be detected Molecular biology experiments typically require detection of extremely small amounts of a particular substance

Autoradiography Autoradiography is a means of detecting radioactive compounds with a photographic emulsion Preferred emulsion is x-ray film DNA is separated on a gel and radiolabeled Gel is placed in contact with x-ray film for hours or days Radioactive emissions from the labeled DNA expose the film Developed film shows dark bands

Autoradiography Analysis Relative quantity of radioactivity can be assessed looking at the developed film More precise measurements are made using a densitometer Area under peaks on a tracing by a scanner Proportional to darkness of the bands on autoradiogram

Phosphorimaging This technique is more accurate in quantifying amount of radioactivity in a substance Response to radioactivity is much more linear Place gel with radioactive bands in contact with a phosphorimager plate Plate absorbs b electrons that excite molecules on the plate which remain excited until plate is scanned Molecular excitation is monitored by a detector

Liquid Scintillation Counting Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting Remove the radioactive material (band from gel) to a vial containing scintillation fluid Fluid contains a fluor that fluoresces when hit with radioactive emissions Acts to convert invisible radioactivity into visible light

Nonradioactive Tracers Newer nonradioactive tracers now rival older radioactive tracers in sensitivity These tracers do not have hazards: Health exposure Handling Disposal Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest

Detecting Nucleic Acids With a Nonradioactive Probe

5.3 Using Nucleic Acid Hybridization Hybridization is the ability of one single-stranded nucleic acid to form a double helix with another single strand of complementary base sequence Previous discussion focused on colony and plaque hybridization This section looks at techniques for isolated nucleic acids

Southern Blots: Identifying Specific DNA Fragments Digests of genomic DNA are separated on a gel The separated pieces are transferred to filter (nitrocellulose) by diffusion, or more recently by electrophoresing the DNA onto the filter The filter is then treated with alkali to denature the DNA, resulting ssDNA binds to the filter A labeled cDNA probe that is complementary to the DNA of interest is then applied to the filter A positive band should be detectable where hybridization between the probe and DNA occurred

Southern Blots The probe hybridizes and a band is generated corresponding to the DNA fragment of interest Visualize bands with x-ray film or autoradiography Multiple bands can lead to several interpretations Multiple genes Several restriction sites in the gene

DNA Fingerprinting and DNA Typing Southern blots are used in forensic labs to identify individuals from DNA-containing materials Minisatellite DNA is a sequence of bases repeated several times, also called a DNA fingerprint Individuals differ in the pattern of repeats of the basic sequence The difference is large enough that 2 people have only a remote chance of having exactly the same pattern of repeats

DNA Fingerprinting Process is a Southern blot Cut the DNA under study with restriction enzyme Ideally cut on either side of minisatellite but not inside Run the digested DNA on a gel and blot Probe with labeled minisatellite DNA and image Note that real samples result in very complex patterns

Forensic Uses of DNA Fingerprinting and DNA Typing While people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashion Can be used to establish parentage Potential to identify criminals Remove innocent people from suspicion Actual pattern has so many bands they can smear together indistinguishably Forensics uses probes for just a single locus Set of probes gives a set of simple patterns

In Situ Hybridization: Locating Genes in Chromosomes Labeled probes can be used to hybridize to chromosomes and reveal which chromosome contains the gene of interest Spread chromosomes from a cell Partially denature DNA creating single-stranded regions to hybridize to labeled probe Stain chromosomes and detect presence of label on particular chromosome Probe can be detected with a fluorescent antibody in a technique called fluorescence in situ hybridization (FISH)

Immunoblots Immunoblots (also called Western blots) use a similar process to Southern blots Electrophoresis of proteins Blot the proteins from the gel to a membrane Detect the protein using antibody or antiserum to the target protein Labeled secondary antibody is used to bind the first antibody for visualization and to increase the signal

Summary Labeled DNA (or RNA) probes can be used to hybridize to DNAs of the same or very similar sequence on a Southern blot DNA fingerprinting can be used as a forensic tool or to test parentage In situ hybridization can be used to locate genes or other specific DNA sequences on whole chromosomes Proteins can be detected and quantified in a complex mixture using Western blots

5.4 DNA Sequencing Sanger, Maxam, and Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNA Modern DNA sequencing is based on the Sanger method and uses dideoxy nucleotides to terminate DNA synthesis The process yields a series of DNA fragments whose size is measured by electrophoresis The last base in each fragment is known as that dideoxy nucleotide was used to terminate the reaction Ordering the fragments by size tells the base sequence of the DNA

Sanger Method of DNA Sequencing

Automated DNA Sequencing Manual sequencing is powerful but slow Automated sequencing uses dideoxynucleotides tagged with different fluorescent molecules Products of each dideoxynucleotide will fluoresce a different color Four reactions are completed, then mixed together and run out on one lane of a gel

High Throughput Sequencing Once an organism’s genome sequence is known, very rapid sequencing techniques can be applied to sequence the genome of another member of the same species Produces relatively short reads or contiguous sequences (25-35bp or 200-300bp, depending on the method) that can easily be pieced together if a reference sequence is available

High Throughput Sequencing Pyrosequencing is one example that is an automated system with the advantages of speed and accuracy - nucleotides are added one by one and the incorporation of a nucleotide is detected by a release of pyrophosphate, which leads to a flash of light Another method (Illumina company) starts by attaching short pieces of DNA to a solid surface, amplifying each DNA in a tiny patch on the surface, then sequencing the patches together by extending them one nucleotide at a time using fluorescent chain-terminating nucleotides, whose fluoresce reveals their identity

Restriction Mapping Prior to the start of large-scale sequencing preliminary work is done to locate landmarks A map based on physical characteristics is called a physical map If restriction sites are the only map features then a restriction map has been prepared

Restriction Map Example Consider a 1.6 kb piece of DNA as an example Cut separate samples of the original 1.6 kb fragment with different restriction enzymes Separate the digests on an agarose gel to determine the size of pieces from each digest Can also use same digest to find the orientation of an insert cloned into a vector

Southern Blots and Restriction Mapping

Summary Physical maps tell about the spatial arrangement of physical “landmarks” such as restriction sites In restriction mapping cut the DNA in question with 2 or more restriction enzymes in separate reactions Measure the sizes of the resulting fragments Cut each with another restriction enzyme and measure size of subfragments by gel electrophoresis Sizes permit location of some restriction sites relative to others Improve process by Southern blotting fragments and hybridizing them to labeled fragments from another restriction enzyme to reveal overlaps

5.5 Protein Engineering With Cloned Genes: Site-Directed Mutagenesis Cloned genes permit biochemical microsurgery on proteins Specific bases in a gene may be changed Amino acids at specific sites in the protein product may be altered as a result Effects of those changes on protein function can be observed

PCR-based Site-Directed Mutagenesis

Summary Using cloned genes, one can introduce changes that may alter the amino acid sequence of the corresponding protein products Mutagenized DNA can be made with: Double-stranded DNA Two complementary mutagenic primers PCR Digest the PCR product to remove wild-type DNA Cells can be transformed with mutagenized DNA

5.6 Mapping and Quantifying Transcripts In the field of molecular biology mapping (locating start and end) and quantifying (how much transcript exists at a set time) transcripts are common procedures Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel Techniques that are specific for the sequence of interest are important

Northern Blots Northern blots detect RNA Example: You have cloned a cDNA Question: How actively is the corresponding gene expressed in different tissues? Answer: Find out using a Northern Blot Obtain RNA from different tissues Run RNA on agarose gel and blot to membrane Hybridize to a labeled cDNA probe Northern plot tells abundance of the transcript Quantify using densitometer

S1 Mapping Use S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a given time Label a ssDNA probe that can only hybridize to transcript of interest Probe must span the sequence start to finish After hybridization, treat with S1 nuclease which degrades ssDNA and RNA Transcript protects part of the probe from degradation Size of protected area can be measured by gel electrophoresis

S1 Mapping the 5’ End

S1 Mapping the 3’ End

Summary A Northern blot is similar to a Southern blot but is a method used for detection of RNA In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative RNase mapping is a variation on SI mapping that uses an RNA probe and RNase

Primer Extension Schematic Primer extension works to determine the 5’-end of a transcript to one-nucleotide accuracy Start with in vivo transcription, harvest cellular RNA containing desired transcript Hybridize labeled oligonucleotide [18nt] (primer) Reverse transcriptase extends the primer to the 5’-end of transcript Denature the RNA-DNA hybrid and run the mix on a high-resolution DNA gel Can estimate transcript concentration also - Specificity of this method is due to complementarity between primer and transcript - S1 mapping will give similar results but with limits: S1 will “nibble” ends of RNA-DNA hybrid Also can “nibble” A-T rich regions that have melted Might not completely digest single-stranded regions

Run-Off Transcription A good assay to measure the rate of in vitro transcription DNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription region Transcribe the truncated fragment in vitro using labeled nucleotides, as polymerase reaches truncation it “runs off” the end Measure length of run-off transcript compared to location of restriction site at 3’-end of truncated gene

Schematic of the G-Less Cassette Assay A variation of the run-off technique in which a stretch of nucleotides lacking guanines is inserted into the nontemplate strand just downstream of the promoter Transcribe altered template in vitro with CTP, ATP and UTP one of which is labeled, but no GTP Transcription will stop when the first G is required resulting in an aborted transcript of predictable size Separate transcripts on a gel and measure transcription activity with autoradiography - The stronger the promoter, the greater number of aborted transcripts is produced

Summary Run-off transcription is a means of checking efficiency and accuracy of in vitro transcription Gene is truncated in the middle and transcribed in vitro in presence of labeled nucleotides RNA polymerase runs off the end making an incomplete transcript Size of run-off transcript locates transcription start site Amount of transcript reflects efficiency of transcription In G-less cassette transcription, a promoter is fused to dsDNA cassette lacking Gs in nontemplate strand Construct is transcribed in vitro in absence of of GTP Transcription aborts at end of cassette for a predictable size band on a gel

5.7 Measuring Transcription Rates in Vivo Primer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a given time These techniques do not really reveal the rate of transcript synthesis as concentration involves both: Transcript synthesis Transcript degradation

Nuclear Run-On Transcription The idea of this assay is to isolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo RNA polymerase that has already initiated transcription will “run-on” or continue to elongate the same RNA chains Effective as initiation of new RNA chains in isolated nuclei does not generally occur Results will show transcription rates and an idea of which genes are transcribed

Nuclear Run-On Transcription Diagram Identification of labeled run-on transcripts is best done by dot blotting Spot denatured DNAs on a filter Hybridize to labeled run-on RNA Identify the RNA by DNA to which it hybridizes Conditions of run-on reaction can be manipulated with effects of product can be measured

Reporter Gene Transcription Place a surrogate reporter gene under the control of a specific promoter and measure the accumulation of the product of this reporter gene The reporter genes are carefully chosen to have products very convenient to assay lacZ produces b-galactosidase which has a blue cleavage product cat produces chloramphenicol acetyl transferase (CAT) which inhibits bacterial growth Luciferase produces a chemiluminescent compound that emits light

Measuring Protein Accumulation in Vivo Gene activity can be monitored by measuring the accumulation of protein, the ultimate gene product There are two primary methods of measuring protein accumulation Immunoblotting / Western blotting (discussed earlier) Immunoprecipitation Immunoprecipitation typically uses an antibody that will bind specifically to the protein of interest followed with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge

5.8 Assaying DNA-Protein Interactions Study of DNA-protein interactions is of significant interest to molecular biologists Types of interactions often studied: Protein-DNA binding Which bases of DNA interact with a protein

Filter Binding Filter binding is used to measure DNA-protein interaction and based on the fact that double-stranded DNA will not bind by itself to a filter, but a protein-DNA complex will Double-stranded DNA can be labeled and mixed with protein Assay protein-DNA binding by measuring the amount of label retained on the filter

Nitrocellulose Filter-Binding Assay dsDNA is labeled and mixed with protein Pour dsDNA through a nitrocellulose filter Measure amount of radioactivity that passed through filter and retained on filter

Gel Mobility Shift DNA moves through a gel faster when it is not bound to protein Gel shift assays detect interaction between protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein

Footprinting Footprinting detects protein-DNA interaction and will show where a target lies on DNA and which bases are involved in protein binding Three methods are very popular: DNase footprinting Dimethylsulfate footprinting Hydroxyl radical footprinting

DNase Footprinting Protein binding to DNA covers the binding site and protects from attack by DNase End label DNA, 1 strand only Protein binds DNA Treat complex with DNase I mild conditions for average of 1 cut per molecule Remove protein from DNA, separate strands and run on a high-resolution polyacrylamide gel

DMS Footprinting Starts the same way as DNase footprinting but then methylate with DMS at conditions for 1 methylation per DNA molecule The protein is then dislodged and treated to remove the methylated purines resulting in apurinic sites which breaks the DNA The DNA fragments are then electrophoresed and autoradiograped for detection Dimethylsulfate (DMS) is a methylating agent

Summary Footprinting finds target DNA sequence or binding site of a DNA-binding protein DNase footprinting binds protein to end-labeled DNA target, then attacks DNA-protein complex with DNase DNA fragments are electrophoresed with protein binding site appearing as a gap in the pattern where protein protected DNA from degradation DMS, DNA methylating agent is used to attack the DNA-protein complex Hydroxyl radicals – copper- or iron-containing organometallic complexes generate hydroxyl radicals that break the DNA strands

Chromatin Immunoprecipitation (ChIP) ChIP is a method used to discover whether a given protein is bound to a given gene in chromatin - the DNA-protein complex that is the natural state of the DNAnin a living cell ChIP uses an antibody to precipitate a particular protein in complex with DNA, and PCR to determine whether the protein binds near a particular gene

Chromatin Immunoprecipitation (ChIP)

5.9 Assaying Protein-Protein Interactions Immunoprecipitation uses an antibody that will bind specifically to the protein of interest and, using a low-speed centrifuge, will ‘pull-down’ any proteins associated with the protein of interest The yeast-two-hybrid assay is used to demonstrate binding (even transient) between two proteins The yeast-two-hybrid assay can also be used to fish for unknown proteins that interact with a known protein

The Yeast-Two Hybrid Assay

5.10 Finding RNA Sequences That Interact With Other Molecules SELEX is systematic evolution of ligands by exponential enrichment SELEX is a method to find RNA sequences that interact with other molecules, even proteins RNAs that interact with a target molecule are selected by affinity chromatography Convert to dsDNA and amplify by PCR RNAs are now highly enriched for sequences that bind to the target molecule

Functional SELEX Functional SELEX is a variation where the desired function alters RNA so it can be amplified If desired function is enzymatic, mutagenesis can be introduced into the amplification step to produce variants with higher activity

5.11 Knockouts and Transgenes Probing structures and activities of genes does not answer questions about the role of the gene in the life of the organism Targeted disruption of genes is now possible in several organisms When genes are disrupted in mice the products are called knockout mice Foreign genes, called transgenes, can also be added to an organism, such as a mouse, to create transgenic mice

Stage 1 of the Knockout Mouse Cloned DNA containing the mouse gene to be knocked out is interrupted with another gene that confers resistance to neomycin A thymidine kinase gene is placed outside the target gene Mix engineered mouse DNA with stem cells so interrupted gene will find way into nucleus and homologous recombination will occur between the altered gene and the resident, intact gene These events are rare, many cells will need to be screened using the introduced genes

Making a Knockout Mouse: Stage 1

Stage 2 of the Knockout Mouse Introduce the interrupted gene into a whole mouse Inject engineered cells into a mouse blastocyst Implant the embryo into a surrogate mother who will give birth to chimeric mouse True heterozygote results when chimera mates with a black mouse to produce brown mice, half of which will have interrupted gene

Making a Knockout Mouse: Stage 2

Knockout Results Phenotype may not be obvious in the progeny, but still instructive Other cases can be lethal with the mice dying before birth Intermediate effects are also common and may require monitoring during the life of the mouse

Methods to Generate Transgenic Mice Two methods to generate transgenic mice: 1. Injection of cloned foreign gene into the sperm pronucleus just after fertilization of a mouse egg but before the sperm and egg nuclei have fused to allow for insertion of the foreign DNA into the embryonic cell DNA 2. Injection of cloned foreign DNA into mosue embryonic stem cells, creating transgenic ES cells Both methods produce chimeric mice that must undergo several rounds of breeding and selection to find true transgenic animals

Summary To probe the role of a gene, molecular biologists can perform targeted disruption of the corresponding gene in a mouse and then look for the effects of the mutation in the ‘knockout mouse’ or insert the foreign gene as a transgene in the ‘transgenic mouse’