ANS/PLSS 433: Gene Probes

1. Introduction:

	The production of diagnostic reagents for the diagnosis of disease 
and genetic disorder is a major industry generating billions of dollars 
annually. Diseases cost the US agriculture $5 bn annually, a variety of 
viruses reduce yield by 10% ($2.5 bn). There are no effective chemical 
controls. Disease containment depends on early detection using diagnostic 
reagents and rapid crop breeding to produce resistant genotypes.

Diagnostic reagents have 3 forms:

	a. Enzyme assay systems, eg the PKU test
	b. Antibodies which detect specific proteins, eg crop virus tests.
	c. DNA or RNA gene sequence probes, eg. potato viroid detection.

	We will concentrate on the slice of the market commanded 
by DNA probes for the next few lectures.
 
2. Relevant DNA chemistry: 

	DNA is made of two strands of bases. Each strand is made of bases 
linked together strongly. The two strands bind together with weak bonds 
between complementary bases, A=T and G=C. We can break the weak bonds of 
DNA by boiling in water and thus separate strands. On lowering the 
temperature complementary strands will eventually hybridize to form dsDNA 
again in a sequence specific manner!

3. Labelling gene probes: 

	Relies on the use of purified DNA polymerase, the enzyme which 
synthesizes DNA during cell division. We simply supply DNA, radiolabelled 
bases and DNA polymerase enzyme and incubate at 37oC for 1 hour to generate 
a gene specific probe which darkens X-ray film. Three methods are 
highlighted by our text, Nick translation, primed synthesis and SP6 
promoter.

	A.	Nick translation makes use of the ability of E.coli DNA 
		polymerase to move or translate nicks in the DNA sample. 

	B.	Primed synthesis uses other DNA polymerases and short 
		random primers to cause DNA synthesis in vitro.
 
	C.	SP6 polymerase utilizes an RNA polymerase a promoter and 
		a restriction site to generate RNA probes of identical sizes. 

	 As we will see later all NA probes can be labelled with 
non-radioactive tags which extends shelf life and utility.

4. Non-radioactive labels for probes:

	Probes labelled with radioactivity are good for research but not 
for a diagnostic laboratory where quality control and long shelf life are 
important for standardization. Two basic non radioactive labelling methods 
have been adopted direct and indirect. Both bind an enzyme to DNA which can 
catalyse an enzymatic conversion of colorless substrate to a colored dye 
(blue, black dyes or even fluorescent light can be produced). However 
these probes may not be as sensitive or versatile as radioactive probes.

Summary:

	DNA is made of two strands which are drawn together by weak bonds 
in a reversible reaction we can control by heating the DNA to 93 C. Using 
DNA polymerase and radioactive nucleotides we can label specific DNA 
molecules so we can see them by exposure to X-ray film. We can use the 
hybridization of labelled DNA to detect infections in plant material.

5. Use of Gene probes to detect viroids:

	Viroids are responsible for important diseases of many crops. 
For example potatoes (spindle tuber viroid), chrysanthemums 
(stunt & chlorotic mottle), hops (stunt), citrus (exocortis) and avocado 
(sunblotch). They can decrease vigor and yield up to 90% in severe cases, 
or in apparently unaffected plants 10 %.  Their control is a problem, 
purchasing virus indexed seed (not virus free since not all viruses can 
yet be detected) is the major defense. Crop rotation, using insecticides 
to kill vectors and planting resistant genotypes are only partially 
effective. Obtaining virus indexed seed requires large tissue culture 
efforts or heavy screening of seed. A diagnostic test for stock material 
is very important. Unfortunately antibodies cant detect viroids. Why? 

Unlike viruses viroids are not encapsulated in protein coats, they are 
simply naked nucleic acids! All are similar being single stranded RNA 
molecules of 300-400 bp. Perfect targets for hybridization!
(They appear to be escaped introns which inhibit RNA processing 
from hnRNA to mRNA).

Probe synthesis:

	For a new disease where a viroid is suspected large amounts of 
viroid RNA can be isolated from infected tissues, copied into cDNA by 
reverse transcriptase and cloned in an E.coli plasmid. This plasmid serves 
as a source of DNA for probes labelled as above. 

Test protocol: 

	Test plants are ground in a buffer and the extract clarified by 
centrifugation. The supernatant is dryed onto a nylon paper which binds 
the RNA irreversibly. The bases (A,T,G,C) are sticking up and are 
available for hybridization. The filter binding sites are blocked with a 
solution of cow DNA and protein then radioactive viroid RNA is added to 
the solution covering the filter. After 24 h at 65 C (25 C below Tm) to 
drive hybridization of identical sequences excess unhybridized probe DNA 
is washed off. Positive hybridization is detected by exposure of the 
filter to film, samples containing viroid turn the film black. 

	Finally probes can be used to identify immune plants in breeding 
programs by deliberate infection and use of probe to find those immune 
plants showing no hybridization signal.