南京大学-杨荣武分子生物学课件1

• Transcription will stop when the first G is required resulting in an aborted transcript of predictable size
1. Crosslink Protein-DNA complexes in situ
2. Isolate nuclei and fragment DNA (sonication or digestion)
3. Immunoprecipitate with antibody
against target nuclear protein and reverse crosslinks
Antibodies as probes
secondary antibody (detects primary antibody)
Required in westerns, but not chromatin immunoprecipitation
Primary antibody
Back to the Chromatin Immunoprecipitation Assay (ChIP)
• RNase mapping uses an RNA probe and RNase
Primer Extension
• Primer extension works to determine exactly the 5’-end of a transcript to one-nucleotide accuracy
Primer Extension Schematic
• Start with in vivo transcription, harvest cellular RNA containing desired transcript
• Hybridize labeled oligonucleotide [18nt] (primer)
degrades ssDNA and RNA – Transcript protects part of the probe from
degradation – Size of protected area can be measured by gel
electrophoresis
S1 Mapping the 5’ End
• If there is no specific antibody, then epitope tagging can be employed (FLAG, MYC, HIS)
• An epitope is a portion of a molecule to which an antibody binds
• Used to determine whether a given protein binds to a given DNA sequence in vivo
• Like all protein analysis involving antibodies (including westerns) a specific antibody is required
• Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel
• Techniques that specific for the sequence of interest are important
Several Useful Molecular biological
Techniques
DNA fingerprinting ChIP Mapping and Quantifying Transcripts
DNA Fingerprinting & Typing
Minisatellite: DNA sequence repeats found througout the (human) genome
• Reverse transcriptase extends the primer to the 5’-end of transcript
• Denature the RNA-DNA hybrid and run the mix on a high-resolution DNA gel
• Can estimate transcript concentration also
Northern Blots
• You have cloned a cDNA
– How actively is the corresponding gene expressed in different tissues?
– Find out using a Northern Blot
• Obtain RNA from different tissues • Run RNA on agarose gel and blot to membrane • Hybridize to a labeled cDNA probe
is found by S1 mapping or primer extension • Useful to see effects of promoter mutation on
accuracy and efficiency of transcription
Run-Off Transcription
• DNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription region
• Transcribe the truncated fragment in vitro using labeled nucleotides, as polymerase reaches truncation it “runs off” the end
• Specificity of this method is due to complementarity between primer and transcript
• S1 mapping will give similar results but limits:
– S1 will “nibble” ends of RNA-DNA hybrid – Also can “nibble” A-T rich regions that have melted – Might not completely digest single-stranded regions
4a. Identify protein
components of isolatNA
sequence by PCR, cloning and sequencing
Mapping and Quantifying Transcripts
• Mapping (locating start and end) and quantifying (how much transcript exists at a set time) are common procedures
• Measure length of run-off transcript compared to location of restriction site at 3’-end of truncated gene
G-Less Cassette Assay
• Variation of the run-off technique, instead of cutting the gene with restriction enzyme, insert a stretch of nucleotides lacking guanines in nontemplate strand just downstream of promoter
– Northern plot tells abundance of the transcript
– Quantify using densitometer
S1 Mapping
Use S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a given time
– Label a ssDNA probe that can only hybridize to transcript of interest
– Probe must span the sequence start to finish – After hybridization, treat with S1 nuclease which
S1 Mapping the 3’ End
Summary
• In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript
• Hybridization of the probe to the transcript protects a portion of the probe from digestion by S1 nuclease, specific for single-stranded polynucleotides
• Length of the section of probe protected by the transcript locates the end of the transcript relative to the known location of an end of the probe
• Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative
• As promoter is stronger a greater number of aborted transcripts is produced
Schematic of the G-Less Cassette Assay
• Transcribe altered template in vitro with CTP, ATP and UTP one of which is labeled, but no GTP
Example of DNA Fingerprint
Autoradiography -means of detecting radioactive compounds with a photographic emulsion (x-ray film)
develop film
Chromatin immunoprecipitation
-individuals differ in the pattern of these repeats
DNA fingerprint is really just a Southern Blot.
Agarose gel
Denature &Transfer to
membrane
Hybridize with probe & detect
Run-Off Transcription and G-Less Cassette Transcription
• If want to assess:
– Transcription accuracy – How much of this accurate transcription
• Simpler method is run-off transcription • Can be used after the physiological start site