Cloning by Subtractive Hybridization

Subtractive hybridization is a technique used to isolate a DNA segment that is missing from one particular sample of DNA. Obviously, a second DNA sample that contains the fragment of interest is necessary. Suppose that a hereditary defect is due to the deletion of the DNA for a particular gene. A sample of DNA from the appropriate chromosome of the afflicted individual will lack this particular segment of DNA. To find the missing DNA, the corresponding chromosome from a healthy (wild type) individual is isolated. For example, the dmd gene, for Duchenne muscular dystrophy, is located in the Xp21 band, close to the middle of the short or p-arm of the X-chromosome. Using light microscopy to analyze chromosomal banding patterns, a patient was found who had a deletion large enough that the Xp21 band was missing. Subtractive hybridization of the mutant chromosome with a normal chromosome, allowed the dmd gene to be cloned.

To do subtractive hybridization, both the mutant and wild type DNA samples are cut into fragments of convenient size using a restriction enzyme. Then the two sets of fragments are hybridized together. This will give hybrid molecules for all regions of the DNA except the region of the deletion, which is present only in the wild type chromosome. If a large surplus of mutant DNA is used, all fragments of the wild type chromosome will be hybridized to mutant fragments except the region corresponding to the deletion, which will be left over. The single strands of this lone fragment will have to pair with each other. Thus, we have subtracted out all the segments of DNA that are not wanted.

In practice, some means to obtain the left over "deletion fragment" is required. One approach is to cut the two batches of DNA with different restriction enzymes. If the normal DNA is cut with restriction enzyme 1 and the mutant DNA is cut with restriction enzyme 2, any hybrid molecule will have non-matching ends. If there mutant DNA hybridized with itself, the ends will be matching, and will cut with restriction enzyme 2. If the gene of interest from the normal DNA self-hybridizes, then this will have compatible ends to restriction enzyme 1. This fragment can then be cloned into a vector using restriction enzyme 1. To make the procedure even easier, restriction enzyme 2 could leave a blunt end after cutting. Since blunt ends are so much harder to ligate, only a self-hybrid molecule flanked by the sticky ends of restriction enzyme 2 would be cloned into the vector. Only the self-paired fragment of wild type DNA would have the sticky ends of restriction enzyme 2 (Fig. 22.29).

Subtractive hybridization can also be used to isolate a set of genes that are expressed under particular conditions. Two batches of cells are grown, one under standard conditions and the other under the conditions being investigated. For example, one batch of mouse cells can be grown with all the necessary nutrients, and another set of mouse cells can be grown with only limited nutrients. The total RNA is isolated from both samples, then the mRNA is purified by hybridization to oligo(dT) as

Dmd gene Gene responsible for Duchenne muscular dystrophy

Duchenne muscular dystrophy One of several inherited diseases affecting mucle function subtractive hybridization Technique used to remove unwanted DNA or RNA by hybridization so leaving behind the DNA or RNA molecule of interest

Step 1

Cut with restriction enzyme #1

Fragment 1A Fragment 1C

Fragment 1B

Probe 1

Use probe 1 Make to isolate Probe 2 fragment 1A

Probe 2

Step 2

Cut with restriction enzyme #2

Fragment 2A

Fragment 2C

Fragment 2B

Probe 2

Make

Use probe 2

to isolate p fragment 2B

Probe 3

Step 3

A) Cut with restriction enzyme #1

B) Use probe 3 to isolate fragment 1B

C) make probe 4

Probe 4

FIGURE 22.28 Chromosome Walking

Chromosome walking utilizes overlapping fragments of a particular chromosome to isolate genes upstream and downstream from the original DNA fragment. The first step is to identify the region of the chromosome to which the probe hybridizes. In this example, probe #1 hybridizes to the purple region of the chromosome. When the chromosome is cut with restriction enzyme #1, fragment 1A will hybridize to probe #1 at one end. This allows fragment 1A to be isolated and sequenced and its downstream sequence is used to generate probe #2. To find the next segment of the chromosome, a different restriction enzyme is used. This time probe #2 will hybridize to fragment 2B. Once again the probe recognizes only the first half of this fragment. The downstream sequence of fragment 2B can then be determined, and this information can be used to make probe #3. Next, the chromosome is cut with restriction enzyme #1 again. Now probe #3 will hybridize with fragment 1B, whose downstream sequence can therefore be determined, and another probe, called probe 4 can be made. This procedure can be continued as far as desired, working in either direction.

FIGURE 22.29 Cloning by Subtractive Hybridization

The key to subtractive hybridization is to hybridize all the wild type or "healthy" DNA fragments (pink) with an excess of mutant DNA (purple). In this example, the mutant DNA is digested with restriction enzyme 1 and the wild type DNA is digested with restriction enzyme 2. Both samples are heated to separate the strands, forming a pool of single-stranded fragments. In order to ensure all the wild type DNA is hybridized to mutant DNA and not to itself, a large surplus of mutant DNA is mixed with a small amount of wild type DNA. The DNA is allowed to anneal yielding double-stranded DNA consisting of a mixture of mutant: mutant, mutant: wild type, and rare wild type : wild type molecules. Since the ratio of wild type DNA to mutant DNA was so low, theoretically the only molecules with two wild type strands should be those containing DNA that is missing from the mutant sampleā€”i.e. the gene of interest. Because two different restriction enzymes were originally used to digest the different samples of DNA, these desired DNA molecules are the only ones that can be digested with restriction enzyme 2. This allows them to be cloned and captured.

Subtractive hybridization can also be performed with two samples of mRNA from the same organism grown under different conditions.

described above. The standard sample will contain mRNA from genes expressed under normal nutrient conditions. The experimental sample will contain mRNA from genes only expressed when nutrients are limited. Limited nutrients may stimulate cells to manufacture their own nutrients, thus some mRNAs would be produced in a higher abundance than the other sample.

The basic idea is that the standard mRNA is used to subtract out the corresponding mRNA molecules from the experimental sample. However, two mRNA molecules of the same sequence obviously cannot hybridize together directly. Therefore, the standard mRNA is first converted to the corresponding double-stranded cDNA by reverse transcriptase. The cDNA is bound to a filter and the experimental sample of

Culture grown under experimental . conditions

Extract mrna

Culture grown under standard conditions

Extract mrna

Make cDna

Most mRNA binds

Only mRNA specific to special growth conditions comes trhrough filter

Most mRNA binds

Only mRNA specific to special growth conditions comes trhrough filter

ssfRifiS s^HJ^^ff^

cDNA IS DENATURED AND BOUND TO FILTER

FIGURE 22.30 Subtractive Hybridization Captures mRNA Expressed under Specific Conditions

Two different cultures are grown, one under standard conditions (green) and one under experimental conditions (orange). The mRNA is isolated from each culture. To allow hybridization, one set of mRNA must be converted into double-stranded DNA by reverse transcriptase. The double-stranded cDNA is then denatured and bound to a filter. In this example, cDNA corresponding to the standard mRNA (green) is bound to the filter. The experimental mRNA (orange) is passed through the filter, where it binds to complementary single-stranded DNA from the standard conditions. If a gene is expressed highly under experimental conditions but not expressed (or present only in low amounts) during standard conditions, its mRNA will not be bound as no corresponding cDNA will be present on the filter. In practice, the mRNA that does not hybridize is pooled and then rehybridized to cDNA from the standard conditions. Repeating this step ensures that all the isolated mRNA is truly absent from the standard sample.

mRNA is passed through (Fig. 22.30). Messenger RNA corresponding to genes in the cDNA is retained by hybridization. Only the mRNA from genes that are expressed under the specific conditions of interest remains unbound and passes through the filter. This, in turn can now be converted to cDNA so giving a sample of those genes expressed under the particular conditions chosen.

Vectors may carry promoters and ribosome binding sites to mediate the expression of cloned genes.

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