![5-56
Primer Extension Schematic
• 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](https://image.slidesharecdn.com/moleculartools-241119083658-b19fbc4b/85/Molecular-tools-Lecture-Note-Presentation-ppt-56-320.jpg)
Molecular tools Lecture Note Presentation.ppt
- 1. Molecular Biology Fourth Edition Chapter 5 Molecular Tools for Studying Genes and Gene Activity Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 2. 5-2 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
- 3. 5-3 Gel Electrophoresis • Gel electrophoresis is used to separate different species of: – Nucleic acid – Protein
- 4. 5-4 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
- 5. 5-5 DNA Separation by Agarose Gel Electrophoresis • DNA is negatively charged due to phosphates in its backbone and moves to anode, the positive pole – Small DNA pieces have little frictional drag so move rapidly – Large DNAs have more frictional drag so their mobility is slower – Result distributes DNA according to size • Largest near the top • Smallest near the bottom • DNA is stained with fluorescent dye
- 6. 5-6 DNA Size Estimation • Comparison with standards permits 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 • Same principles apply to RNA separation
- 7. 5-7 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)
- 8. 5-8 Protein Gel Electrophoresis • Separation of proteins is done using a gel made of polyacrylamide (polyacrylamide gel electrophoresis = PAGE) – Treat proteins to denature subunits with detergent such as 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
- 9. 5-9 Summary • DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis • Most common gel used in nucleic acid electrophoresis is agarose • Polyacrylamide is usually used in protein electrophoresis • SDS-PAGE is used to separate polypeptides according to their masses
- 10. 5-10 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 can be done: – (no SDS) uses 2 consecutive gels – Sequential gels with first a pH separation, then separate in a polyacrylamide gel
- 11. 5-11 A Simple 2-D Method • Run samples 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
- 12. 5-12 Two-Dimensional Gel Electrophoresis Details 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
- 13. 5-13 More Two-Dimensional Gel Electrophoresis Details • 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
- 14. 5-14 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 sample loaded onto the column material
- 15. 5-15 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
- 16. 5-16 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, exclude larger ones • Larger substances travel faster through the column
- 17. 5-17 Affinity Chromatography • In 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 the specific binding
- 18. 5-18 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
- 19. 5-19 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
- 20. 5-20 Autoradiography Analysis • Relative quantity of radioactivity can be assessed looking at the developed film • More precise measurements are made using densitometer – Area under peaks on a tracing by a scanner – Proportional to darkness of the bands on autoradiogram
- 21. 5-21 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 electrons that excite molecules on the plate which remain excited until plate is scanned – Molecular excitation is monitored by a detector
- 22. 5-22 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
- 23. 5-23 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
- 24. 5-24 Detecting Nucleic Acids With a Nonradioactive Probe
- 25. 5-25 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
- 26. 5-26 Southern Blots: Identifying Specific DNA Fragments • Digests of genomic DNA are separated on agarose gel • The separated pieces are transferred to filter by diffusion, or more recently by electrophoresing the bands onto the filter • Filter is treated with alkali to denature the DNA, resulting ssDNA binds to the filter • Probe the filter using labeled cDNA
- 27. 5-27 Southern Blots • Probe cDNA 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
- 28. 5-28 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 DNA fingerprint – Individuals differ in the pattern of repeats of the basic sequence – Difference is large enough that 2 people have only a remote chance of having exactly the same pattern
- 29. 5-29 DNA Fingerprinting Process really just a Southern blot •Cut the DNA under study with restriction enzyme – Ideally cut on either side of minisatellite but not inside •Run digest on a gel and blot •Probe with labeled minisatellite DNA and imaged – Real samples result in very complex patterns
- 30. 5-30 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
- 31. 5-31 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)
- 32. 5-32 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 and increase the signal
- 34. 5-34 DNA Sequencing • Sanger, Maxam, 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
- 35. 5-35 Sanger Manual Sequencing Sanger DNA sequencing method uses dideoxy nucleotides to terminate DNA synthesis – The process yields a series of DNA fragments whose size is measured by electrophoresis – 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
- 37. 5-37 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
- 39. 5-39 Restriction Mapping • Prior to 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 • Consider a 1.6 kb piece of DNA as an example
- 40. 5-40 Restriction Map 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
- 42. 5-42 Using Restriction Mapping With an Unknown DNA Sample
- 44. 5-44 Southern Blots and Restriction Mapping
- 45. 5-45 Summary • Physical map tells 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
- 46. 5-46 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 also be altered – Effects of those changes on protein function can be observed • Might investigate the role of phenolic group on tyrosine compared to phenylalanine
- 48. 5-48 Summary • Using cloned genes, can introduce changes at will to alter amino acid sequence of 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
- 49. 5-49 5.4 Mapping and Quantifying Transcripts • Mapping (locating start and end) and quantifying (how much transcript exists at a set time) are common procedures • Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel • Techniques that specific for the sequence of interest are important
- 50. 5-50 Northern Blots • You have cloned a cDNA – How actively is the corresponding gene expressed in different tissues? – 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
- 51. 5-51 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
- 52. 5-52 S1 Mapping the 5’ End
- 53. 5-53 S1 Mapping the 3’ End
- 54. 5-54 Summary • In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript • Hybridization of the probe to the transcript protects a portion of the probe from digestion by S1 nuclease, specific for single-stranded polynucleotides • Length of the section of probe protected by the transcript locates the end of the transcript relative to the known location of an end of the probe • Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative • RNase mapping uses an RNA probe and RNase
- 55. 5-55 Primer Extension • Primer extension works to determine exactly the 5’-end of a transcript to one-nucleotide accuracy • Specificity of this method is due to complementarity between primer and transcript • S1 mapping will give similar results but 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
- 56. 5-56 Primer Extension Schematic • 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
- 57. 5-57 Run-Off Transcription and G- Less Cassette Transcription • If want to assess: – Transcription accuracy – How much of this accurate transcription • Simpler method is run-off transcription • Can be used after the physiological start site is found by S1 mapping or primer extension • Useful to see effects of promoter mutation on accuracy and efficiency of transcription
- 58. 5-58 Run-Off 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
- 59. 5-59 G-Less Cassette Assay • Variation of the run-off technique, instead of cutting the gene with restriction enzyme, insert a stretch of nucleotides lacking guanines in nontemplate strand just downstream of promoter • As promoter is stronger a greater number of aborted transcripts is produced
- 60. 5-60 Schematic of the G-Less Cassette Assay • 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
- 61. 5-61 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
- 62. 5-62 5.5 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
- 63. 5-63 Nuclear Run-On Transcription • Isolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo in a technique called run-on transcription • RNA polymerase that has already initiated transcription will “run-on” or continue to elongate same RNA chains • Effective as initiation of new RNA chains in isolated nuclei does not generally occur
- 64. 5-64 Run-On Analysis • Results will show transcription rates and an idea of which genes are transcribed • 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
- 66. 5-66 Reporter Gene Transcription • Place a surrogate reporter gene under control of a specific promoter, measure accumulation of product of this reporter gene • Reporter genes are carefully chosen to have products very convenient to assay – lacZ produces -galactosidase which has a blue cleavage product – cat produces chloramphenicol acetyl transferase (CAT) which inhibits bacterial growth – Luciferase produces chemiluminescent compound that emits light
- 67. 5-67 Measuring Protein Accumulation in Vivo • Gene activity can be monitored by measuring the accumulation of protein (the ultimate gene product) • Two primary methods of measuring protein accumulation – Immunoblotting / Western blotting – Immunoprecipitation
- 68. 5-68 Immunoprecipitation • Label proteins by growing cells with 35 S- labeled amino acid • Bind protein of interest to an antibody • Precipitate the protein-antibody complex with a secondary antibody complexed to Protein A on resin beads using a low- speed centrifuge • Determine protein level with liquid scintillation counting
- 69. 5-69 5.6 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
- 70. 5-70 Filter Binding Filter binding to measure DNA-protein interaction is 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
- 71. 5-71 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
- 72. 5-72 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
- 73. 5-73 Footprinting • Footprinting shows 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
- 74. 5-74 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
- 75. 5-75 DMS Footprinting • Dimethylsulfate (DMS) is a methylating agent which can fit into DNA nooks and crannies • Starts as DNase, then methylate with DMS at conditions for 1 methylation per DNA molecule
- 76. 5-76 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
- 77. 5-77 5.7 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
- 78. 5-78 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
- 79. 5-79 5.8 Knockouts • 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
- 80. 5-80 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 with altered gene and resident, intact gene • These events are rare, many cells will need to be screened using the introduced genes
- 81. 5-81 Making a Knockout Mouse: Stage 1
- 82. 5-82 Stage 2 of the Knockout Mouse • Introduce the interrupted gene into a whole mouse • Inject engineered cells into a mouse blastocyst • Embryo into a surrogate mother who gives birth to chimeric mouse with patchy coat • True heterozygote results when chimera mates with a black mouse to produce brown mice, half of which will have interrupted gene
- 83. 5-83 Making a Knockout Mousse: Stage 2
- 84. 5-84 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