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5.2 Labeled Tracer Techniques
❖Autoradiography ❖Phosphorimaging ❖Nonradioactive Tracers
Autoradiography
Figure 5.9 Densitometry. An autoradiograph is pictured beneath a densitometer scan of the same film. Notice that the areas under the three peaks of the scan are proportional to the darkness of the corresponding bands on the autoradiograph
Figure 5.16 A Northern blot. Poly(A)+ RNA was isolated from the rat tissues indicated at the top, then equal amounts of RNA from each tissue were electrophoresed and Northern blotted. The RNAs on the blot were hybridized to a labeled probe for the rat glyceraldehyde-3phosphate dehydrogenase (G3PDH) gene, and the blot was then exposed to x-ray film. The bands represent the G3PDH mRNA, and their intensities are indicative of the amounts of this mRNA in each tissue.
❖Phosphorimaging
Figure 5.10 False color phosphorimager scan of an RNA blot. After hybridizing a radioactive probe to an RNA blot and washing away unhybridized probe, the blot was exposed to a phosphorimager plate. The plate collected energy from β-rays from the radioactive probe bound to the RNA bands, then gave up this energy when scanned with a laser. A computer converted this energy into an image in which the colors correspond to radiation intensity according to the following color scale: yellow (lowest) < purple < magenta < light blue < green < dark blue < black (highest).
tandem.
DNA fingerprinting
Figure 5.14 DNA fingerprint. (a) The nine lanes contain DNA from nine unrelated white subjects. Note that no two patterns are identical, especially at the upper end. (b) The two lanes contain DNA from monozygotic twins, so the patterns are identical (although there is more DNA in lane 10 than in lane 11). (Source: G. Vassart et al. Science 235 (6 Feb 1987) p. 683)
1) Southern Blot: Identifying Specific DNA Fragments
2) Northern Blot: Measuring Gene Activity 3) In Situ Hybridization: Locating Genes in Chromosomes
➣ Western Blot: Identifying Specific Proteins
Chapter 5. Molecular Tools for Studying Genes and Gene Activity
Molecular Tools for Studying Genes and Gene Activity 5.1 Molecular Separations 5.2 Labeled Tracers 5.3 Using Nucleic Acid Hybridization 5.4 DNA Sequencing Techniques 5.5 Mapping and Quantifying Transcripts 5.6 Measuring Transcription Rates in Vivo 5.7 Assaying DNA–Protein Interactions 5.8 Knockouts
DNA Typing
2) Northern Blots: Measuring Gene Activity
Suppose you have cloned a cDNA and want to know how actively the corresponding gene (gene X) is transcribed in a number of different tissues of organism Y. You could answer that question in several ways, but the method we describe here will also tell the size and the relative amounts of the mRNA the gene produces.
Single Molecule Sequencing
❖ The Sanger dideoxy method of DNA sequencing
Figure 5.19 A typical sequencing film. The sequence begins CAAAAAACGG. You can probably read the rest of the sequence to the top of the film. (Source: Courtesy Life Technologies, Inc., Gaithersburg, MD.)
3) In Situ Hybridization
One can hybridize labeled probes to whole chromosomes to locate genes or other specific DNA sequences. This type of procedure is called in situ hybridization; if the probe is fluorescently labeled, the technique is called fluorescence in situ hybridization (FISH).
5.1 Molecular Separations
❖Gel Electrophoresis
Agarose Gel Electrophoresis
PAGE (PolyAcrylamide Gel Electrophoresis)
Agarose Gel Electrophoresis
20 ng
(20 mm/910 bp)
11
human muscle glycogen phosphorylase gene
5.4 DNA Sequencing Techniques
❖The Sanger Chain-Termination Sequencing Method ➣Automated DNA sequencing
❖ Maxam–Gilbert Sequencing Method ❖ Pyrosequencing (454) ❖ Solexa (Illumina) ❖ SOLid (ABI)
➣Standard agarose gel electrophoresis
➣Pulsed-field gel electrophoresis(PFGE)
The bands represent chromosomes having sizes ranging from 0.2 Mb (at bottom) to 2.2 Mb (at top). Original gel is about 13 cm wide by 12.5 cm long. The 16 visible bands represent 16 chromosomes Pulsed-field gel electrophoresis of yeast chromosomes
➣Automated DNA sequencing
❖ Maxam–Gilbert Sequencing Method The MaxamGilbert sequencing method follows a very different strategy. Instead of synthesizing DNA ithesis reactions with chain terminators, this method starts with fulllength, end-labeled DNA and cleaves it with basespecific reagents. The Maxam–Gilbert method allows us to break an end-labeled DNA strand at specific bases using base-specific reagents. We can then electrophoresis the labeled fragments in a highresolution polyacrylamide gel and detect them by autoradiography. This procedure orders the fragments by size and therefore tells us the