PRINCIPLES OF CROP PRODUCTIONABT-320

(3 CREDIT HOURS)

LECTURE 15

MAPPING GENES ON SPECIFIC CHROMOSOMESSOMATIC CELL HYBRIDSIN SITU HYBRIDIZATIONTRANSPOSON TAGGING

GENETIC LINKAGE MAPPINGGENOMIC LIBRARIES

COLONY HYBRIDIZATIONPLAQUE HYBRIDIZATION

CHROMOSOMAL WALKING AND JUMPINGCLEAVING DNA WITH RESTRICTION ENZYMES

GEL ELECTROPHORESISBLOTTING TECHNIQUES

MAPPING GENES ON SPECIFIC CHROMOSOMES

One of the most important applications of nucleic acid hybridization is construction of genetic maps. This helps in determining the chromosomal location of individual genes. There are three commonly used methods that differ in the degree of resolution they afford viz somatic cell hybrids, hybridization and genetic linkage mapping.

SOMATIC CELL HYBRIDSSomatic cell hybrids are prepared by fusing together human and rodent cells using reagents, such as polyethylene glycol. The reagent helps in membrane fusion. The resulting hybrid cells are then grown in culture medium containing selective agents which allow only the hybrid cells to grow. In hybrids produced using human and rodent cells the rodent chromosomes are retained while the human chromosomes are lost. This random chromosome loss occurs during continued culture and shows no apparent selectivity for individual human chromosomes. Thus it is possible to produce a panel of hybrid cells containing different combinations of human chromosomes. Genomic DNA is prepared from panels of such hybrids and used in hybridization analysis. The human probe to be mapped is hybridized with such hybrids. By determining which of the panel of hybrids shows a hybridization signal, and correlating this information with the karyotype of the cells, it is possible to deduce which chromosome carries the test gene.

IN SITU HYBRIDIZATION

• In this technique a labelled DNA probe is hybridized to metaphase chromosome. Metaphase chromosomes are liberated from cells and bound to the surface of the slide. The DNA within the chromosomes is denatured in such a way that the individual chromosomes remain visually identifiable. The probe is labelled, either by incorporation of a 3H-nucleotide or non-isotopically. The chromosomes are incubated with the labelled probe for many hours and, after hybridization, the excess unbound probe is washed away and the annealed probe detected.

• If radiolabelled probe used then the slides are autoradiographed. Deposited silver grain after autoradiography will lie in the approximate area at which the probe bounds to the chromosome. The accuracy of mapping by this technique is dependent on the size of the grains which, in the case of small chromosomes, can cover a significant portion of the total chromosome length.

IN SITU HYBRIDIZATION

• If the probe is labelled non-isotopically then it is possible to determine the precise position of hybridization with much greater accuracy. The signal is detected using a fluorescent molecule thus is called fluorescent in situ hybridization (FISH). It is more sensitive than radiolabelling and can be used to give a fairly precise position of hybridization.

• Thus FISH is particularly useful for determining the respective locations of two markers on the same chromosome. The two probes are labelled with different immunologically labeled adducts and a single hybridization reaction is performed. The two probes are then detected using fluorochromes which emit at different wavelengths when excited by uv light such as rhodamine (red fluorescence) and fluorescein (green fluorescence). This technique has been used for probes which are separated by few hundred kilobases of DNA on same chromosome.

FISH is used where gene mapping is done with in-situ hybridization. It uses a non-radioactive labeling system involving biotin attached. When viewed under special filters the specific sites of DNA probe can be seen on a chromosome.

The principle of non-radioactive detection methods for in-situ hybridization

TRANSPOSON TAGGING

• Isolating a eukaryotic gene by transposon tagging involves inserting into target DNA, fractionating fragments of the latter on southern blots and hybridizing the transposon with a probe. Cleaving out the transposon, together with flanking DNA, reveals the adjoining sequences of a gene. The probe are made for these adjoining sequences to find the fragment having gene of interest.

• Presence of transposon is confirmed by altered phenotype. This method was first employed to isolate white locus in Drosophila. The mutants with white-apricot eyes harboured a copia retrotransposon as well as accompanying white locus.

GENETIC LINKAGE MAPPINGThis form of mapping is used to construct genetic linkage maps of individual chromosomes and also to localize genes involved in genetic disease. Genetic linkage analysis relies on an ability to estimate the frequency of crossing over (recombination) that occurs between homologous chromosomes during meiosis. The closer two genetic markers are to each other then the lower is the chance that they will be separated during meiosis.

GENOMIC LIBRARYThe complete set of cloned fragments of DNA of an organism is known as genomic library. This is also called gene library. Such a library contains all or most of the genes present in the cell or the organism. It is difficult to identify a particular gene from an organism and then clone it. Hence the organism’s entire genome as DNA fragments is cloned and the clone containing the sequence of interest is identified.Cloning experiments can be divided into two types:

1. Those where the cloned DNA segment is of unknown composition and function and

2. Those where the cloned DNA segment is of known composition and function.

In the first approach the entire cellular DNA has to be fragmented into clonable size by either digestion with restriction endonucleases or mechanical sheering.

GENOMIC LIBRARY

• These experiments with randomly cloned fragments are known as ‘shot gun cloning experiments’.

• The chromosomal DNA of the organism of interest is isolated. It is then treated with a known restriction enzyme to obtain DNA fragments of clonable size. The fragments are cloned using appropriate cloning vectors in any organisms like bacteria, virus or yeast. For identification, a marker gene is inserted while multiplying in vector. Hybridization of such genes can be carried out by two major techniques viz. colony hybridization and plaque hybridization depending on the type of vector used.

COLONY HYBRIDIZATIONOnce a genomic library or cDNA library is available, gene sequence can be isolated by colony hybridization techniques. It involves replica plating the colonies formed by transformed cell into nitrocellulose filter and their in situ hybridization with a radioactive probe. Its procedure is as follows:

1. The cloned yeast colonies, bacterial colonies or phage plaques to be tested are transferred from the culture plate on to a nitrocellulose filter paper by replica plating.

2. The filter with the colony replicas is treated with NaOH to lyse the cells/phages and to denature DNA.

3. The filter is then baked to fix the DNA.4. It is then treated with a radioactive 32P labelled single stranded DNA

probe that is complementary to the ssDNA of interest.5. The filter is washed to remove the unbound excess probe, dried and

then autoradiographed.

COLONY HYBRIDIZATION

6. The DNA fragments hybridized with the radioactive probe can be detected on the autoradiograph film.

7. This indicates that the cells in the corresponding colonies contain the desired gene. The colony is taken out from the master culture plate for mass culture.

Colony hybridization to identify the clones containing the DNA of interest

PLAQUE HYBRIDIZATIONThis technique is applicable where a chimeric phage particle is carrying the gene of interest. In such a situation, a bacterial lawn is infected with a mixture of chimeric phage particles (i.e., the library) and a large number of plaques develop overnight. These plaques can be treated just like the colonies in a colony hybridization to identify and isolate the chimeric phage particle carrying the gene of interest. Two new techniques have shown their potential in gene isolation and characterization. They are chromosome walking and chromosome jumping.

CHROMOSOME WALKINGIdentification of fragments with overlapping sequences may be a key to the reconstruction or characterization of large chromosome regions. This is achieved by the technique popularly described as chromosome walking.It involves the following steps:

1. From the genomic library a clone of interest identified by a probe is selected.

2. One end of the clone is subcloned.3. The subcloned fragment of the selected clone may be hybridized with

other clones in the library and a second clone hybridizing with the subclone of the first clone is identified due to presence of overlapping region.

4. The end of the second is then subcloned and used for hybridization with other clones. This helps in identifying a third clone having overlapping region with the subcloned end of the second clone.

CHROMOSOME WALKING

5. Identified third clone is also subcloned and hybridized with clones in the same manner and the procedure may be continued.

6. Restriction map of each selected clone is prepared and compared to know the region of overlapping, so that identification of few overlapping restriction sites resembles the walking along the chromosome or along a long chromosome segment.

Regions of chromosomes approaching 1000 kb have been mapped following the above technique. Restriction maps of entire chromosomes can be prepared in this manner following the technique of chromosome walking.

CHROMOSOME JUMPING

In order to bring the molecular marker close to the gene of interest, chromosome jumping approach has been utilized. This technique can be described in the following steps:

1. The distance of ‘jumps’ or ‘hopsize’ (e.g. 100kb or 200kb) is determined.2. Genomic DNA molecules in the range of selected size are selected

through pulsed-field gel electrophoresis.3. For circularization of DNA segments, ligation between two ends of each

long linear molecule is allowed using T4 ligase.4. These circular DNA circles are digested with EcoR1.5. Phage vector Ch3A lac, is also cut with EcoR1 and used for cloning small

DNA fragments.

CHROMOSOME JUMPING

6. Such cloned DNA fragments represent the jumping library, which can be plated on a bacterial host and screened through the technique of plaque hybridization.

The technique of chromosome jumping helps in narrowing the gap between the gene and available molecular markers. After several cycles of chromosome jumping followed by cloning the regions proximal to the gene, it will be possible to approach very close to the desired gene. The gene for cystic fibrosis has recently been isolated using this technique.

CLEAVING DNA WITH RESTRICTION ENZYMES

• After locating gene of interest in colony or on chromosome, the DNA is extracted and cleaved with enzymes. Most restriction enzymes cleave DNA molecules in a site specific manner. The genomic DNA from an individual organism is digested with either a single or more restriction enzymes. This results in fragmentation of DNA molecules with variation in length. These fragments are known as restriction fragment length polymorphism or RFLPs. Since restriction enzymes cut DNA in a sequence specific manner, every homologous DNA molecule will be cut at exactly the same site. This allows to isolate large quantities of specific DNA fragments for subcloning and isolating the DNA sequence of interest.

• The sizes of the restriction fragments can be determined by polyacrylamide or agarose gel electrophoresis. It also helps in separation of various RFLPs.

GEL ELECTROPHORESIS• Nucleic acid molecules can be distinguished by the differences in their

chain length.• DNA and RNA have a net negative charge. At neutral pH therefore, these

molecules migrate towards the positive terminal when placed in an electric field. This process of electrophoresis is performed in an agarose or polyacrylamide gel. Nucleic acids are loaded into slots in the gel and allowed to migrate towards the positive terminal. The pores in the gel act to sieve the molecules. So that the mobility of a particular nucleic acid species depends on its length. All the molecules of a particular size move at approximately the same rate through the gel, forming a band which gradually increases in width during electrophoresis because of diffusion. The size of the molecules can be estimated by running DNA molecules of known size in a parallel track on the gel, and determining their migration position relative to the species of the unknown size.

GEL ELECTROPHORESIS• Polyacrylamide has a smaller average pore size than agarose and so is

effective at separating molecules of 10-1000 nucleotides in length (1000 nucleotides is termed 1 kilobase or 1 kb), with very high resolution. The larger pores in agarose allows it to resolve much bigger molecules, up to 100 kb in length. In pulsed-field gel electrophoresis (PFGE) the electrical field across the gel is repeatedly switched back and forth between two or more directions. This allows very long DNA molecules, several million base pairs in length, to be separated, since very big molecules change direction less rapidly as they move through the agarose gel.

• After gel electrophoresis, the nucleic acid molecules must be detected. They can be visualized directly by including ethidium bromide in the polyacrylamide or agarose gel. This dye binds to double-stranded nucleic acids by intercalating between adjacent base pairs, and emits a red fluorescence when exposed to uv radiation. As little as 5-10 ng of DNA can be detected. If the nucleic acid molecules are radioactively labelled, then individual resolved species can be detected by autoradiography.

BLOTTING TECHNIQUES

These are:

1. Analysis of DNA by Southern Blotting2. Analysis of RNA by Northern Blot Hybridization3. Analysis of Protein by Western Blot Hybridization4. Dot Blots and Slot Blots

ANALYSIS OF DNA BY SOUTHERN BLOTTING

• In 1975, E. M. Southern invented a procedure to identify the location of genes and other DNA sequences on restriction fragments separated by gel electrophoresis. The essential feature of this technique is the transfer of the DNA molecules separated by gel electrophoresis to a nitrocellulose or nylon membrane.

• The DNA is denatured either prior to or during transfer by placing the gel in an alkaline solution. After transfer is complete, the DNA is immobilized on the membrane by drying or UV-induced cross-linking to the filter. A radioactive DNA (probe) containing the sequence of interest is then hybridized or annealed with the immobilized DNA on the membrane. The probe will anneal to form a double helix only with complementary DNA molecules on the membrane. Nonannealed probe is then washed off the membrane, and the washed membrane is exposed to X-ray film that detects the presence of the radioactivity in the bound probe. After the autoradiogram is developed, the dark bands show the position(s) of DNA sequences that have hybridized with the probe.

ANALYSIS OF DNA BY SOUTHERN BLOTTING

The ability to transfer DNA restriction fragments or other DNA molecules that have been separated by gel electrophoresis to nitrocellulose or nylon membranes for hybridization studies and other types of analyses has proven to be extremely useful. Such transfers of DNA to membranes are called Southern blots after the name of its inventor.

ANALYSIS OF RNA BY NORTHERN BLOT HYBRIDIZATION

• As DNA molecules can be transferred from agarose gels to nitrocellulose or nylon membranes for hybridization studies, RNA molecules can also be separated by agarose gel electrophoresis similarly and transferred for analyses analyzed. Such RNA transfers are used routinely in molecular genetics laboratories and are called northern blots as it is the mirror image of the Southern blotting technique.

• The northern blot procedure is essentially identical to that used for Southern blot transfers however, degradation by RNA molecules are more sensitive to degradation by RNases. Thus, gloves must be worn at all times to prevent contamination of solutions with RNases on one’s fingers. In addition, all glassware must be baked overnight to inactivate RNases, and all chemical reagents must be baked or be highly purified to ensure that they are RNase-free. RNase molecules are extremely stable enzymes; they are not inactivated by boiling or other treatments that would totally destroy most enzymes.

ANALYSIS OF RNA BY NORTHERN BLOT HYBRIDIZATION

• Most RNA molecules contain considerable secondary structure. Therefore, they must be kept denatured during electrophoresis to separate them on the basis of size. Denaturation is accomplished by adding formaldehyde or some other chemical denaturant to the loading buffer and the electrophoresis running buffer. After transfer to an appropriate membrane, the RNA blot is hybridized to either RNA or DNA probes just as with Southern blots.

• Northern blot hybridization is extremely useful in the study of gene expression. They can be used to determine whether a particular gene is transcribed in all tissues of an organism or only certain tissues. They can also be used to study the temporal pattern of expression of individual genes during growth and development. Major disadvantage with northern blot hybridization is that it only measures the accumulation of RNA transcripts, but explains nothing about why the observed accumulation occurs.

ANALYSIS OF PROTEIN BY WESTERN BLOT TECHNIQUE

• Polyacrylamide gel electrophoresis has been used as an important tool for the separation and characterization of proteins.

• Since many functional proteins are composed of two or more subunits, individual polypeptides are separated by electrophoresis in the presence of the detergent sodium dodecyl sulfate (SDS), which denatures the proteins.

• After electrophoresis, the proteins are commonly detected by treating with Coomassie blue or silver stain. However, the separated polypeptides in the gel can also be transferred to a nitrocellulose membrane, and individual proteins can be detected by using specific antibodies. This transfer of proteins from acrylamide gels to nitrocellulose membranes is called western blotting and is performed by using an electric current hence called electroblotting.

ANALYSIS OF PROTEIN BY WESTERN BLOT TECHNIQUE

After transfer, a specific protein of interest is identified by placing the membrane with the immobilized proteins in a solution containing an antibody to the protein. Non-bound antibodies are then washed off the membrane, and the presence of the initial (‘primary’) antibody is detected by placing the membrane in a solution containing a ‘secondary’ antibody. This ‘secondary’ antibody reacts with immunoglobulins (the group of protein containing all antibodies) in general. The secondary antibody is conjugated to either a radioactive isotope (permitting autoradiography) or an enzyme that produces a visible product when the proper substrate is added. The western blot procedure is a very powerful tool for identifying and characterizing specific gene products.

THE END