Ch' 5 Molecular Tools

1
Ch. 5 Molecular Tools
2
(No Transcript)
3
(No Transcript)
4
Molecular Separations

• Ion-Exchange chromatography
• Gel-Filtration chromatography

5
Southern Blots
-transfer of DNA onto a medium on which
hybridization is convenient
Hybridization- complementary base pairing between
DNA (RNA) from different sources
6
The Transfer or Blotting Process
Paper or filter/membranes must bind nucleic
acids nitrocellulose, PVDF (Polyvinylidene
Fluoride). NOTE Denaturation step.
7
Detection or Visualization
-Probe nucleic acid with label radioactive or
chemiluminescent
-Hybridize with labeled probe.
8
(No Transcript)
9
DNA Fingerprinting Typing
Minisatellite DNA sequence repeats found
througout the (human) genome
-individuals differ in the pattern of these
repeats
10
DNA fingerprint is really just a Southern Blot.
Agarose gel
Denature Transfer to membrane
Hybridize with probe detect
11
Example of DNA Fingerprint
12
Labeling of (probe) DNA
1. Incorporation of a radioactive atom (32P)
End label using 32P-ATP and DNA kinase
2. Incorporation of a radioactive nucleotide into
the DNA. 32P-dNTP
Use PCR
3. Chemiluminescent methods
13
(No Transcript)
14
Autoradiography
-means of detecting radioactive compounds with a
photographic emulsion (x-ray film)
develop
film
15
In Situ Hybridization Locating Genes in
Chromosomes

• Labeled probes can be used to hybridize to
  chromosomes and reveal which chromosome contains
  the gene of interest
• Spread chromosomes from a cell
• Partially denature DNA creating single-stranded
  regions to hybridize to labeled probe
• Stain chromosomes and detect presence of label on
  particular chromosome
• Probe can be detected with a fluorescent antibody
  in a technique called fluorescence in situ
  hybridization (FISH)

16
5.4 Mapping and Quantifying Transcripts

• Mapping (locating start and end) and quantifying
  (how much transcript exists at a set time) are
  common procedures
• 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

17
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
• Northern plot tells abundance of the transcript
• Quantify using densitometer

18
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
  degrades ssDNA and RNA
• Transcript protects part of the probe from
  degradation
• Size of protected area can be measured by gel
  electrophoresis

19
S1 Mapping the 5 End
20
S1 Mapping the 3 End
21
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
• RNase mapping uses an RNA probe and RNase

22
Primer Extension

• Primer extension works to determine exactly the
  5-end of a transcript to one-nucleotide accuracy
• 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

23
Primer Extension Schematic

• Start with in vivo transcription, harvest
  cellular RNA containing desired transcript
• Hybridize labeled oligonucleotide 18nt (primer)
• 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

24
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 is
  found by S1 mapping or primer extension
• Useful to see effects of promoter mutation on
  accuracy and efficiency of transcription

25
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
• Measure length of run-off transcript compared to
  location of restriction site at 3-end of
  truncated gene

26
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
• As promoter is stronger a greater number of
  aborted transcripts is produced

27
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
• Transcription will stop when the first G is
  required resulting in an aborted transcript of
  predictable size
• Separate transcripts on a gel and measure
  transcription activity with autoradiography

28
Summary

• Run-off transcription is a means of checking
  efficiency and accuracy of in vitro transcription
• Gene is truncated in the middle and transcribed
  in vitro in presence of labeled nucleotides
• RNA polymerase runs off the end making an
  incomplete transcript
• Size of run-off transcript locates transcription
  start site
• Amount of transcript reflects efficiency of
  transcription
• In G-less cassette transcription, a promoter is
  fused to dsDNA cassette lacking Gs in nontemplate
  strand
• Construct is transcribed in vitro in absence of
  of GTP
• Transcription aborts at end of cassette for a
  predictable size band on a gel

29
5.5 Measuring Transcription Rates in Vivo

• Primer extension, S1 mapping and Northern
  blotting will determine the concentration of
  specific transcripts at a given time
• These techniques do not really reveal the rate of
  transcript synthesis as concentration involves
  both
• Transcript synthesis
• Transcript degradation

30
Nuclear Run-On Transcription

• Isolate nuclei from cells, allow them to extend
  in vitro the transcripts already started in vivo
  in a technique called run-on transcription
• RNA polymerase that has already initiated
  transcription will run-on or continue to
  elongate same RNA chains
• Effective as initiation of new RNA chains in
  isolated nuclei does not generally occur

31
Run-On Analysis

• Results will show transcription rates and an idea
  of which genes are transcribed
• Identification of labeled run-on transcripts is
  best done by dot blotting
• Spot denatured DNAs on a filter
• Hybridize to labeled run-on RNA
• Identify the RNA by DNA to which it hybridizes
• Conditions of run-on reaction can be manipulated
  with effects of product can be measured

32
Nuclear Run-On Transcription Diagram
33
Reporter Genes

• Antibiotic Resistance used for determining
  which cells contain the vector
• Promoter strength Antibiotic resistance can be
  quantitated (Camr)
• LacZ
• Fluorescence Proteins

34
ß-galactosidase (blue/white screening, promoter
strength)

• Lac operon
• Hydrolyzes lactose, and lactose analogs such as
  X-gal
• The digestion of X-gal produces a blue dye
• Can be used to screen for insert
• Can be used to determine promoter strength

35
(No Transcript)
36
Measuring DNA-Protein Interactions
37
Nitrocellulose filter binding assay
38
Gel mobility shift assay
1. Labeled DNA DNA-protein complexes are
subjected to gel electrophoresis (agarose or
polyacrylamide)
2. Detect DNA (labeled or ethidium bromide)
39
DNA Footprinting DNase Method
40
Electrophoresis after Dnase treatment
DNA bands visualized (autoradiography)
41
Actual DNase footprint
42
DNA Footprinting DMS Method
(dimethylsulfate)
43
DMS treatment
1. DNA is end-labeled protein added
2. DNA-protein complex is methylated with DMS
such that only one methlyation event occurs per
DNA molecule
3. Piperidine-removes methylated purines
(apurinic sites) ? breaks in the DNA at these
sites
4. DNA fragments electrophoresed visualized by
autoradiography
44
DMS (small molecule) method more subtle than DNase
In DMS method each band ends next to a nucleotide
that was methylated (unprotected by protein)
Note band becoming darker ? binding of protein
distorts DNA d-helix such that the base
corresponding to this band is more vulnerable to
methylation
45
Actual DMS Footprint
46
(Yeast) Two Hybrid

• Transcriptional factor contains both a DNA
  binding domain and an activation domain
• System splits these two domains into separate
  proteins
• Bait protein is attached to one (activator
  domain)
• Target library (fish) is constructed attached to
  the other (DNA-binding domain)
• Interaction between bait and target brings
  activator and DNA-binding domains together ?
  leading to transcription

47
(No Transcript)