Molecular Tools for Studying Genes and Gene Activity Molecular Biology Lecture 7 Chapter 5 Molecular Tools for Studying Genes and Gene Activity Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

ATGAGAGGATCG GGATCCGCATGC---------AAGCTT 6) (6 points) For your independent research project, your supervisor is asking you to clone the coding region of gene X (see below) in the bacterial expression vector pQE30 using the polymerase chain reaction (PCR). Write the sequence of two oligonucleotides that will allow you to clone the coding sequence in the vector. The recombinant protein must be as short as possible. Note: The coding sequence must be in frame with the ATG of the vector. The start and stop codons of gene X are underlined. Coding sequence of gene X GTCGATCAAT ATGGAACATG TTTACTCCAA ACCACCGCAC ACCAATTATG GAAACCAAGC CGGAAAAGAA TTCCGGTGGA GAGCGAAAAA AAAGGATTCC GAATCGTGAA CTGCCAAAAA CATTTTGAAG CCAACGATTC CGACGTCATC CTCGCCACCC TAGCTAAATC AGGCACCACT TGGTTAAAAG CTCTTCTCTT TGCTCTCATT CACCGACACA AGTTCCCAGT TTCTGGCAAG CATCCTCTTC TGAAACAGCA GTAGCAGCGT TTAAAGGGAA GTTTATT Oligo #1 5’ ___________________________________________ Oligo #2 5’ ___________________________________________ BamH1 = GGATCC HindIII = AAGCTT RBS 6Xhis ATGAGAGGATCG GGATCCGCATGC---------AAGCTT RBS 6Xhis ATGAGAGGATCG ACGGATCCGCATGC---------AAGCTT

Lecture outline Gel electrophoresis Nucleic acids Proteins Radioactive tracers Southern blot Western blot Genomic or cDNA library screening DNA hybridization Antibody screening

Gel Electrophoresis 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

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

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

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

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

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 densitometer Area under peaks on a tracing by a scanner Proportional to darkness of the bands on autoradiogram

Detecting Nucleic Acids With a Nonradioactive Probe

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

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 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

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

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

Western Blots

Genomic library construction (very complex library) Dna extraction Digestion with restriction endonuclease Ligation in vector Selection cDNA library construction (simplified library) Extract mRNA Reverse transcription to make cDNA

DNA hybridization

Selection of clone of interest DNA hybridization Must have a probe to screen the library Antibody screening Must have antibodies specific for your protein

Antibody screening

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

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

Sanger 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

Automated DNA Sequencing