1. Chapt. 5 Molecular Tools for Studying Genes and Gene Activity Learning outcomes : Be able to state basic principles and processes of selected tools Demonstrate ability to interpret experiments with these tools Important Figs: 2, 4, 6, 7, 8, 11*, 12, 15, 17, 18*, 20, 26 Review Q: 1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 21; Analyt Q: 1, 2 5-1

2. 5-2 5.1 Molecular Separations Mixtures of proteins or nucleic acids made during molecular biological procedures –A protein purified from crude cellular extract –A particular nucleic acid molecule made in a reaction needs to be purified Gel electrophoresis and chromatography widely used techniques for separating specific molecules from mixture

3. 5-3 DNA Gel Electrophoresis Melted agarose poured into mold with comb Comb “teeth” form slots (wells) in solidified agarose DNA samples placed in wells Electric current through gel separates molecules Stain DNA with EtBr (Recall BL 261, BL 415) Fig. 1

4. 5-4 DNA Separation by Agarose Gel Electrophoresis DNA negatively charged (phosphates) moves to anode, positive pole –Small DNA pieces move rapidly (little frictional drag) –Large DNAs move slower –DNA distributes according to size: Largest near top Smallest near bottom DNA visualized by staining with fluorescent dye (EtBr) (Fig. 2a) + -

5. 5-5 DNA Size Estimation - Compare with standards Mobility of fragments plotted v. log of molecular weight (log number of base pairs) – linear for smaller ones Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation Special techniques for huge DNA Similar principles apply to RNA separation (denature) Fig. 2b

6. 5-6 Protein Gel Electrophoresis + - Separate proteins on polyacrylamide gel (polyacrylamide gel electrophoresis = PAGE) –denature protein subunits with detergent SDS SDS coats polypeptides with negative charges so all move to anode Masks natural charges of proteins, so all move relative to mass not charge –Smaller proteins move faster toward the anode; stain proteins; compare to protein standards Fig. 4

7. 5-7 Ion-Exchange Chromatography Protein sample loaded; buffer passed over resin Ionic strength of buffer increases, samples flowing through column are collected Samples tested for presence of protein of interest (A 280, protein gel, enzyme activity; Fig. 6) Resin separates substances by charge

8. 5-8 Gel Filtration Chromatography Gel filtration uses columns filled with porous resins: let in smaller substances, exclude larger ones (ex. sephadex); elute with only one buffer Protein size - basis of physical separation Larger substances travel faster through the column Fig. 7

9. 5-9 Affinity Chromatography Resin contains a substance to which molecule of interest has strong and specific affinity Molecule binds to resin having affinity reagent –Molecule of interest is retained –Other molecules flow through without binding –Molecule of interest eluted from column using specific solution that disrupts specific binding Ex. His-tagged proteins bind Ni+ resin; eluted with increasing imidazole concentrations

10. 5-10 5.2 Labeled Tracers - often means “radioactive” Detect very small quantities of proteins, DNA, RNA Autoradiography detects radioactive compounds with photographic emulsion –x-ray film –Radiolabeled DNA on gel –Gel in contact with x-ray film –Radioactive emissions from labeled DNA expose film –Developed film shows dark bands

11. 5-11 Autoradiography Analysis Relative quantity of radioactivity assessed from developed film More precise measurements are made with densitometer –Area under peaks by scanner (Fig. 9) –Proportional to darkness of bands on autoradiogram

12. 5-12 Nonradioactive Tracers Nonradioactive tracers rival radioactive tracers in sensitivity These tracers do not have hazards: –Health exposure –Handling –Disposal Increased sensitivity because multiplier effect of enzyme coupled to probe for molecule of interest

13. 5-13 Detecting Nucleic Acids With Nonradioactive Probe Biotinylated DNA probe; detect with avidin-alkaline phosphatase; Other probes labeled with Digoxigenin; detect with antibody coupled to alkaline phosphatase ( BL 427 ) Fig. 11

14. 5-14 5.3 Using Nucleic Acid Hybridization Hybridization - single-stranded nucleic acid forms double helix with another single strand of complementary base sequence (RNA, DNA) Previous colony and plaque hybridization Detect with nucleic acid probes Techniques for isolated nucleic acids

15. 5-15 Southern Blots Separate DNA on gel; denature, transfer to filter Probe DNA hybridizes; Band corresponds to DNA fragment of interest Visualize bands with X-ray film, nonradioactive method Multiple bands lead to several interpretations –Multiple genes –Several restriction sites in gene Fig. 12

16. 5-16 DNA Fingerprinting and DNA Typing Southern blots in forensic labs identify individuals from DNA-containing materials (Jeffreys et al., 1986) Minisatellite DNA - sequence of bases repeated several times, also called DNA fingerprint –Individuals differ in repeats of basic sequence – –Difference large enough that 2 people have only remote chance of exactly same pattern Other repeated DNA sequences (VNTR) used: people only two bands; probe for several loci for statistical significance

17. 5-17 DNA Fingerprinting Like Southern blot Cut DNA with restriction enzyme –Ideally cut either side of minisatellite, not inside Run digest on gel, blot Probe with minisatellite DNA Real samples very complex (Fig. 13)

18. Tandem repeat polymorphisms Fig 2.25: Different numbers of repeats can be distinguished by PCR using primers to flanking DNA Or by Southern with cutting DNA

19. 5-19 Forensic Uses of DNA Fingerprinting People different DNA fingerprints: pattern inherited Mendelian fashion –Establish parentage; identify criminals; clear innocent people Actual pattern many bands; smear together indistinguishably –Forensics uses several probes for single loci; places where many alleles are possible (SSR, VNTR) –Set of probes gives set of simple patterns Fig. 15

20. 5-20 In Situ Hybridization: Locating Genes in Chromosomes jjjjjjjjj Fig. 16 Labeled probes hybridize to specific genes on chromosomes: –Spread chromosomes from cell arrested in metaphase –Partially denature DNA, creates single-stranded regions, –hybridizes to labeled probe –Stain chromosomes, detect presence of label on chromosome Probe can be detected with fluorescent antibody in technique called fluorescence in situ hybridization (FISH)

21. 5-21 Immunoblots (Western blots) – can quantify Similar process to Southern blots, but proteins: –Electrophoresis of proteins –Blot proteins from gel to membrane –Detect protein using primary antibody to target protein –Labeled secondary antibody binds first antibody and increases signal (can be nonradioactive) Fig. 17

22. 5-22 DNA Sequencing Modern DNA sequencing based on Sanger method: Dideoxy nucleotides terminate DNA synthesis –4 reactions, lots of 3 dNTPs,1 ddNTP in each: ddTTP reaction has some dTTP, lots of dATP, dCTP, dGTP –Series of DNA fragments whose size is accurately measured by electrophoresis (high % PAG) –Last base in each fragment is known, that dideoxy nucleotide used to terminate the reaction –Ordering fragments by size tells base sequence of DNA that was synthesized

23. 5-23 Sanger dideoxy DNA Sequencing (is DNA synthesis) High resolution PAG gels distinguish fragments that differ in size by 1 base; Fig. 18

24. 5-24 Automated dideoxy DNA Sequencing Fig. 20 Dideoxynucleotides tagged with different fluorescent molecules: Products of each dideoxynucleotide termination fluoresce different color Four reactions completed, mixed, run on one lane of gel Detector reads colors and calculates sequence

25. 5-25 5.4 Mapping and Quantifying Transcripts Mapping (locating start and end); Quantifying (how much transcript at a set time) Transcripts often not uniform terminator -: continuum of species smeared on gel Techniques specific for sequence of interest Nuclease S1 mapping locates 5’ and 3’ ends (later)

26. 5-26 Northern Blots quantify RNA With cloned cDNA, ask: –How is gene expressed in different tissues? Run RNA from tissues on agarose gel, blot to membrane Hybridize to labeled cDNA or other probe –Northern tells abundance of transcript –Northern tells size of transcript –Quantify using densitometer (Fig. 26)

27. 5-27 5.5 Reporter Gene Transcription Place (surrogate) reporter gene under control of specific promoter, measure accumulation of product of reporter gene -> protein = gene expression Reporter genes chosen to have products convenient to assay –lacZ produces  -galactosidase, makes blue cleavage product with Xgal (BCIG) substrate –cat produces chloramphenicol acetyl transferase (CAT) which inactivates Chloramphenicol –Luciferase (luc) produces chemiluminescent compound that emits light (detect luminometer)

28. 5-28 Measuring Protein Accumulation in Vivo Gene activity in different tissues, different times, monitored by measuring accumulation of protein (the ultimate gene product) Two methods measure protein accumulation –Immunoblotting / Western blotting uses antibodies to detect proteins separated on gels –Immunoprecipitation uses antibodies to precipitate protein of interest from solution, test what other proteins, DNA or RNA co-precipitate.

29. Review questions 1.Illustrate principle of DNA gel electrophoresis; indicate comparative mobilities of DNAs with 150, 600, 1200 bp. 2.Compare process of Southern blot and RNA blot in terms of process, and what information can be provided. 12. Diagram imaginary Sanger sequencing gel, and provide DNA sequence. 21. Describe use of reporter gene to measure strength of a promoter. 5-29