Bio 423 Lecture 2

BB211: Cell and Molecular Biology Dr Eve Lutz Department of Bioscience

Recombinant DNA technology: Lecture 2 Southern/Northern Analysis Background reading: Reference for this lecture please read the following: Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

Analysing complex nucleic acid mixtures (DNA or RNA) The total cellular DNA of an organism (genome) or the cellular content of RNA is complex mixtures of different nucleic acid sequences. Restriction digest of a complex genome can generate millions of specific restriction fragments and there can be several fragments of exactly the same size which will not be separated from each other by electrophoresis. Techniques have been devised to identify specific nucleic acids in these complex mixtures • •

Southern blotting - DNA Northern blotting - RNA

These techniques are not to be confused with Western blotting, which is used to analyze PROTEINS which have been immobilized

on nitrocellulose/nylon filters. Proteins which have been separated by polyacrylamide gel electrophoresis (PAGE) are transferred to nitrocellulose/nylon filters and the filter is probed with antibodies to detect the specific protein - similar to the method used for expression library screening.

Southern blotting This technique, devised by Ed Southern in 1975, is a commonly used method for the identification of DNA fragments that are complementary to a know DNA sequence. Southern hybridization, also called Southern blotting, allows a comparison between the genome of a particular organism and that of an available gene or gene fragment (the probe). It can tell us whether an organism contains a particular gene, and provide information about the organization and restriction map of that gene. In Southern blotting, chromosomal DNA is isolated from the organism of interest, and digested to completion with a restriction endonuclease enzyme. The restriction fragments are then subjected to electrophoresis on an agarose gel, which separates the fragments on the basis of size. DNA fragments in the gel are denatured (i.e. separated into single strands) using an alkaline solution. The next step is to transfer fragments from the gel onto nitrocellulose filter or nylon membrane. This can be performed by electrotransfer (electrophoresing the DNA out of the gel and onto a nitrocellulose filter), but is more typically performed by simple capillary action.

Fig 7-32, Lodish et al (4th ed.) In this system, the denatured gel is placed onto sheet(s) of moist filter paper and immersed in a buffer reservoir. A nitrocellulose membrane is laid over the gel, and a number of dry filter papers are placed on top of the membrane. By capillary action, buffer moves up through the gel, drawn by the dry filter paper. It carries the single-stranded DNA with it, and when the DNA reaches the nitrocellulose it binds to it and is immobilized in the same position relative to where it had migrated in the gel. The DNA is bound irreversibly to the filter/membrane by baking at high temperature (nitrocellulose) or cross-linking through exposure to UV light (nylon). The final step is to immerse the membrane in a solution containing the probe - either a DNA (cDNA clone, genomic fragment, and oligonucleotide) or RNA probe can be used. This is DNA hybridization - in other words the target DNA and the probe DNA/RNA form a 'hybrid' because they are complementary sequences and so can bind to each other. The probe is usually radioactively labeled with 32P (high energy β-particle emitter), often by removal of the 5' phosphate of the probe with alkaline phosphatase, and replacement with a radiolabeled phosphate using γ-[32P] ATP and polynucleotide kinase. The membrane is washed to remove non-specifically bound probe (see washing & stringency conditions), and is then exposed to X-ray film - a process called autoradiography. At positions where the probe is bound, β-

emissions from the probe cause the X-ray film to blacken. This allows the identification of the sizes and the number of fragments of chromosomal genes with strong similarity to the gene or gene fragment used as a probe. The principle of Southern blotting

What Southern blotting can tell us: 1. Whether a particular gene is present and how many copies

are present in the genome of an organism 2. The degree of similarity between the chromosomal gene and the probe sequence 3. Whether recognition sites for particular restriction endonucleases are present in the gene. By performing the digestion with different endonucleases, or with combinations of endonucleases, it is possible to obtain a restriction map of the gene i.e. an idea of the restriction enzyme sites in and around the gene- which will assist in attempts to clone the gene.

4. Whether re-arrangements have occurred during the cloning

process

An example of a Southern blot of human genomic clones hybridised with a 1.5 kb cDNA probe

The size of this gene is ~117 kb and the mRNA transcript is ~4.8 kb - the gene is organised into 13 exons. Because of the large size of the gene, several genomic clones, from YAC, PAC and cosmid libraries, were isolated by hybridization screening with the cDNA probe. In order to produce a partial restriction map of the gene, the genomic clone cDNAs were digested with XhoI, HindIII, EcoRI and BamHI individually, separated by agarose gel electrophoresis and blotted onto nylon membranes. a) shows the YAC clone digests (lanes 1-4), b) shows 2 different PAC clones and their digests (PAC clone 1: lanes 1-4; PAC clone 2: lanes 6-9) and c) shows 7 cosmid clones, each digested with BamHI. A cDNA probe encoding the whole open reading frame (ORF) was labeled with 32P and used to hybridize with these. Positive bands correspond to DNA fragments containing exon sequences (a cDNA

probe will hybridize to fragments containing exon sequences). Lanes containing the same size fragment for the same restriction digest indicate that some clones overlap each other (contain the same region of DNA). The sizes of the molecular weight markers are shown alongside each blot.

Northern blotting Northern blotting is a simple extension of Southern blotting and derives its name from the earlier technique. It is used to detect cellular RNA rather than DNA. Initially, it was thought that RNA would not bind efficiently to nitrocellulose, and other modified materials were synthesized for use as a membrane. However, it was then shown that when RNA was denatured, that it would also bind efficiently to nitrocellulose. This means that the RNA has to be unfolded into a linear strand before it will bind efficiently to nitrocellulose. Chemicals such as formaldehyde and methylmercuric hydroxide can be used to denature the RNA breaking down hydrogen bonding structure in the molecule. Alkali is not used to denature the RNA - since RNA is degraded under alkaline conditions. Isolating RNA RNA is extracted from the cells of interest - but precautions must be taken to avoid degradation of the single-stranded RNA by ribonuclease (RNase), which is found on the skin and on glassware. Wear gloves, use specially treated plastics and glassware to avoid accidentally introducing ribonuclease to extraction prep. Addition of diethylpyrocabonate (DEPC) inhibits ribonuclease activity and baking at high temperature destroys ribonuclease activity (only useful for treating heat resistant equipment, such as glassware). Remember: it is important to maintain the integrity of RNA molecules

RNA blots are most usually probed with cDNA fragments, but if a single stranded probe (such as an oligonucleotide or a transcribed RNA) is used - MAKE SURE IT IS COMPLEMENTARY TO THE RNA SEQUENCE of the gene of interest. What Northern blotting can tell us 1. Differential expression patterns of a particular gene a) In which tissues it is expressed b) If it is expressed during certain stages of development c) If expression changes under differing conditions/treatments of the cell 2. The quantity of the mRNA present •

Blots can be quantitated accurately by radioactive counting

3. Whether a genomic DNA probe has regions that are transcribed An example of a Northern blot exposed to film:

Fig 7-33, Lodish et al (4th ed.) Relative abundance of mRNA for β-globin in leukaemia cells that are growing (UN) and in cells that have been induced to undergo differentiation. The β-globin mRNA is produced when the cells begin to differentiate. Both Southerns & Northerns are exquisitely sensitive Both Southern and Northern blots can detect very small amounts of nucleic acids. Blots with both RNA and DNA from different organisms (zoo blots) can inform us on the conservation of genes among different organisms. website last updated 20/2/03 Back to Recombinant DNA Technology Lecture List