1. Molecular Tools for Studying Genes and Gene Activity
Chapter 5
Molecular Tools for Studying Genes and Gene Activity

2. _ +
In neutral pH buffer
Friction force

3. logarithmic

4. For the DNAs in the size ranges ~Mb

5. Pre-stained protein markers

9. Labeled Tracers Autoradiography b-emitters: 3H, 14C, 35S, 32P
Radioactive DNA
fragments
Autoradiography
b-emitters: 3H, 14C, 35S, 32P

11. Greater linearity than autoradiography
Phosphorimaging
Greater linearity than autoradiography
Radioactive sample
B-ray
Molecules (ground state) in Image plate
Excited molecules in image plate
Energy released from excited molecules
by a laser bean from phosphorimager
Detected by computerized detector
Liquid Scintillation Counting

12. Non-radioactive Tracers

13. Using Nucleic Acid Hybridization
Southern Blots:
Identifying Specific DNA Fragments
(Edward Southern--the pioneer)
Band number > 1 ???

14. DNA Fingerprinting and DNA Typing
Alec Jeffreys (1985): minisatellite (repeated) sequence in a-globin gene
also in the whole human genome
Minisatellite sequence has no RE site for HaeIII

15. Monozygotic twins
Unrelated Caucasians

16. Forensic Uses of DNA Fingerprinting and DNA Typing

17. Northern Blots: Measuring Gene Activity
Poly(A)+ RNA: from rat tissues
Probe: G3PDH (glyceraldehyde-3-phosphate dehydrogenase)

18. In situ Hybridization: Locating genes in chromosomes
FISH:
Fluorescence
in situ hybridization

19. DNA Sequencing 1975 Frederick Sanger Alan Maxam Walter Gilbert
3 billion bases of
human genome
The Sanger Chain-termination
Sequencing Method
Using didexoy nucleotides

20. CAAAAAACGG

21. Automated DNA sequencing

22. Maxam-Gilbert
Sequencing
(Dimethyl sulfate:
guanine)
(piperidine)

27. Protein Engineering of Cloned Genes: Site-directed Mutagenesis
Change of protein function by change of the gene sequence

28. Step 1 T A G G
PCR C G C
Step 2
PCR C
Step 3 G C
PCR G C
PCR with

29. Mapping and Quantifying Transcripts
Mapping: locating the starting and stopping points of transcripts
Quantifying: measuring how much of a transcript exists at a certain time

30. S1 Mapping for 5’-end of a transcript3’

31. S1 Mapping for 3’-end of a transcript5’

33. Primer Extension: To map the end of a transcript
with one-nucleotide accuracy
S1 nuclease tends nibble a bit
on the RNA-DNA hybrid, and
A-T-rich regions can melt transiently
TTCGACTGACAGT

34. Run-off Transcription
To check the efficiency and accuracy of in vitro transcription
(can be used to assay the promoter before or after mutation)

35. Run-off Transcription

36. Measuring Transcription Rates In Vivo
Nuclear Run-on Transcription
Initiation of new RNA chains in isolated nuclei does not generally occur
(using heparin to inhibit free RNA polymerase and prevent re-initiation)

37. Reporter Gene Transcription
CAT:
Chloramphenicol (CAM)
acetyl transferase
CAM:
Protein synthesis inhibitor
acetylation by CAT
Loss inhibitor activity
Other reporter enzymes:
b-galatosidase
luciferase

38. Assaying DNA-Protein Interactions
Filter Binding
Nitrocellulose membrane binds proteins and ssDNA but not dsDNA
dsDNA-protien complex do bind to NC

39. Gel Mobility Shift Assay (Electrophoresis Mobility Shift Assay, EMSA)

40. DNase Footprinting
protein

41. * * DMS Footprinting Methylating agent: Dimethylsulfate
1,4 no protein added

42. Knockouts

43. Brown:
dominant