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

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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

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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

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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)

+

-

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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 DNAReal samples

very complex

(Fig. 13)

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

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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

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In Situ Hybridization: Locating Genes in Chromosomes

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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)

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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

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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

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Sanger dideoxy DNA Sequencing(is DNA synthesis)

High resolution PAG gels distinguish fragments that differ in size by 1 base;

Fig. 18

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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

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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)

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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)

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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)

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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.

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.

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