Southern and Northern blotting

Transcription

1 Southern and Northern blotting

2 Comparison of Southern, Northern, and Western analyses of Gene X

3 SOUTHERN BLOTTING The technique was developed by E.M. Southern in The Southern blot is used to detect the presence of a particular piece of DNA in a sample. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

4 Southern hybridization Transfer buffer

5 Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Isotope Non-isotope Hybridization Post-hybridization washing Signal detection

6 1) DNA Fragmentation The DNA sample is digested with one or more restriction enzymes. Nuclease hydrolyze the bonds connect bases within the strand. If large amount of restriction fragments faint smears rather than discrete bands Same or nearly the same length migrate together during electrophoresis

7 2) Gel Electrophoresis Separate the fragments according to size to be detected using ethidium bromide. Smaller fragments are migrating faster than larger fragments.

8 Gel Treatment Double stranded DNA denatured using NaOH (base). separating it into single DNA strands for later hybridization to the single stranded probe. If too large, treated with HCL (acid) to depurinate & break it into smaller fragment & neutralized before proceed.

9 Gel treatment Acid treatment 0.2 N HCl solution Denaturation NaOH solution Neutralization Tris-Cl buffer (ph8.0)

10 Depurination Optional Occurs before neutralization When DNA fragments is > 15 kilobases, it is too hard to be transferred to filter The gel is treated with dilute acid (0.2 M HCl for 15 minutes) depurinate DNA fragment into smaller pieces and promote higher efficiency transfer to filter.

11 Neutralization DNA is placed into an alkaline solution containing 0.5mM NaOH to denature the dsdna into ssdna and neutralize the acid in previous step. Function: (1) improve binding of the vely charged DNA to +vely charged filter (2) ssdna strands for hybridization (3) destroy any remaining RNA present in the sample Only ssdna can be transferred to filter

12 3. Blotting Exert pressure evenly to a gel to ensure even contact between gel and membrane Transfer is done by capillary action which draws buffer (binds ssdna) up onto the membrane The binding of DNA to membrane is due to ion exchange interactions

13 Southern transfer Measure gel and set up transfer assembly: Wick in tray with 20x SSC Gel Nitrocellulose or Nylon filters (soaked in H 2 O and 20x SSC) 3MM Whatman filter paper Paper towels Weight

14 Blotting Transferred the gel onto a membrane which is a sheet of nitrocellulose filter or nylon membrane to carry out blotting. Enable the DNA fragments accessible by the label probes required for analysis by autoradiography or other detection methods depend on your probes.

15 Blotting Gel is placed on the filter paper dipped in a reservoir containing transfer buffer. The membrane is laid on top of the gel wit paper towels placing on top of the membrane. Buffer lift up by capillary action to move the DNA from the reservoir to the membrane.

16 Blot permanently Binding of the DNA to the membrane is weak after blotting (unstable). Easily to washed off during washing. A) Treated the DNA fragments with UV light B) Oven baked it around 80 C.

17 4) Pre Hybridization- Blocking To block non specific sites. To ensure the specificity of the binding of the probe to the sample DNA.

18 5) Probe Labeling The probe DNA is labeled so that it can be detected. A)Radioactive labeling B)Tagging with non radioactive probes

19 6) Hybridization Process of forming a double-stranded DNA molecule between a singlestranded DNA probe and a singlestranded target patient DNA. The reactions are specific where the probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

20 7) Post Hybridization- Washing Excess unbound probe is washed away with buffers containing NaCl and detergent.

21 8) Detection Labeled probe bound to the membrane is detected by autoradiography. Reveals the position of DNA fragment to which the probe hybridized. Autoradiograph is an image on an x-ray film which will visualized the discrete bands directly caused by beta emissions from the 32 P radioactivelabeled probe. Hybridization of the probe to the sample DNA fragment on the membrane indicates that the fragment contains DNA sequence that is complementary to the probe.

22 Autoradiography Exposure to x-ray film

23 SOUTHERN BLOTTING The key to this method is hybridization. Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

24 Step 3. DNA Denaturation Eliminate hydrogen bonds with sodium hydroxide (NaOH) A C T T G A T G A A C T

25 Step 4. Transfer DNA to Membrane Two methods for transferring DNA to a membrane capillary electrophoretic

26 Other Applications DNA fingerprinting RFLP of VNTRs Dot or slot blot Colony or plaque lifts Microarray analysis

27 Other Applications DNA fingerprinting RFLP of VNTRs Dot or slot blot Colony or plaque lifts Microarray analysis

28 Other Applications DNA fingerprinting RFLP of VNTRs Dot or slot blot Colony or plaque lifts Microarray analysis

29 Other Applications DNA fingerprinting RFLP of VNTRs Dot or slot blot Colony or plaque lifts Gene Expression

30 Other Applications Microarray technology

31 Northern Blotting Almost the same with Southern blot. Northern blotting is used for detecting RNA fragments. Southern blotting is used for detecting DNA fragments. Technique for detecting specific RNA separate by electrophoresis using RNA probe.

32 Northern blotting Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.

33 Application Advantages Disadvantages Northern blotting is able to detect small changes in gene expression that microarrays cannot. Thousand of genes visualized at a time with microarray, while northern blotting is usually looking at one or small number of genes

34 The flow chart of Northern hybridization Prepare RNA samples and run RNA gel Northern transfer Probe preparation Prehybridization Isotope Non-isotope Hybridization Post-hybridization washing Signal detection

35 An example of Northern blotting Northern blot RNA gel 28 S 18 S

36 Comparison of Southern and Northern Blot Molecule detected Gel electrophoresis Gel pretreatment Blotting method Probes Detection system Southern blotting DNA Agarose gel Depurination, denaturation, and neutralization Capillary transfer DNA Radioactive or nonradioactive Autoradiography Chemiluminescent Colorimetric Northern blotting mrna Formaldehyde agarose gel - Capillary transfer cdna, Radioactive or nonradioactive Autoradiography Chemiluminescent Colorimetric