1. Molecular Tools for Studying Genes and Gene Activity
Molecular Biology
Lecture 7
Chapter 5
Molecular Tools for Studying Genes and Gene Activity
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2. 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

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

4. Gel Electrophoresis
Gel electrophoresis is used to separate different species of:
Nucleic acid
Protein

5. 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

6. 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

7. 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

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. 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. 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

11. 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

12. 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

13. Detecting Nucleic Acids With a Nonradioactive Probe

14. 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

15. 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

16. 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

17. 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

18. 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

19. Western Blots

20. 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

21. DNA hybridization

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

23. Antibody screening

24. 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

25. 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

26. Sanger DNA Sequencing

27. 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

28. Automated DNA Sequencing