ANS/PLSS 433: Molecular Markers

Introduction:  

	We saw last week that labelled DNA could serve as a probe for 
viroids in a simple positive or negative screen of total DNA and RNA from 
leaf samples. With some modification (see below) the same type of screen 
can even be used to detect whole foreign chromosomes introduced by 
interspecific crosses in breeding programs, certain infectious diseases 
or food spoiling organisms.

1.  Contaminant detection:

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.

	Viruses, Bacteria and other microorganisms can be detected and typed 
in foodstuffs, plant tissues or humans with a very similar approach to 
viroid detection. But, the bacterial or virus DNA must be exposed by 
efficient lysis. Alkali and detergent are often used (like our solution 2 
in the practical) to both lyse cells and denature the DNA (hydrogen bonds 
break in alkali). Microorganism detection by gene probes competes for 
market share with antibody probes (which recognize characteristic proteins 
associated with the microorganisms by their shape). Each approach has 
advantages.

   Gene probes                       Antibody probes.

1. Radioactive                       Non-radioactive
2. 24-72h per screen                 6-12h per screen
3. Uses dirty samples                Needs pure proteins
4. Less false negatives              False negatives and positives
5. Doesnt distinguish living cells   May distinguish living cells

	PCR probes combine the advantages and disadvantages of gene probes 
with non-radioactive detection and theoretically unlimited sensitivity. 
Primers which hybridize to an enterotoxin gene for example will amplify a 
1 kb fragment 240 fold (1012 fold), enough DNA that it can be seen directly 
as follows:

	DNA Fragments are separated by size by Gel Electrophoresis. 
DNA can be pushed by electricity through an agarose gel. DNA is positively 
charged and migrates away from the negative electrode and toward the 
positive. Large fragments move more slowly because they bump into the gel 
molecules more often than small ones.  Fragments of identical size migrate 
identical distances and form a band. DNA bands can be seen in the gel as 
DNA binds ethidium bromide, a chemical which fluoresces orange under UV 
light.  However the PCR method is prone to both false positives and 
negatives.

2.  Southern Blotting:

	Southern blotting increases the diagnostic potential of DNA probe 
hybridization but requires several additional steps to the viroid protocol.

	1. Purified DNA is cut with a sequence specific restriction 
		endonuclease. The fragments produced from different genes 
		are of different lengths, the fragments produced by 
		identical genes are identical lengths. 

	2. Fragments are separated by size by Gel Electrophoresis. DNA can 
		be pushed by electricity through an agarose gel. DNA is 
		positively charged and migrates away from the negative 
		electrode and toward the positive. Large fragments move 
		more slowly because they bump into the gel molecules more 
		often than small ones.

	3. DNA is denatured, whilst still in the gel the two strands 
		separated to make them available for hybridization. The 
		strands are separated by soaking the gel in alkali.

	4. DNA is Blotted onto a nylon filter with pores too small for DNA 
		to pass but large enough for water and salt molecules to 
		pass. The nett result is a filter paper replica of the gel 
		DNA fragment pattern.

	5. Probe hybridization as before for viroid detection 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 at a band of a characteristic size for that species 
		of viroid. 

	Southern hybridization may be less sensitive and slower than PCR 
but it is not as prone to false positives or negatives.  

3.  Southern hybridization and Restriction Fragment Length Polymorphism

Introduction: 

	Southern hybridization as a technique is used to increase the 
specificity of gene probes. In the practical we will run our first 
electrophoresis gel, an important part of Southern hybridization. We will 
have a photograph of the DNA band positions in the gel, which reflect the 
size of the DNA molecules. When we blot the gel we get a permanent record 
which can be hybridized with gene probes. In Lab, we will digest our 
DNA with restriction enzymes and run another gel of the products.

RFLP:

	Restriction Fragment Length Polymorphism (or RFLP) detected by 
Southern hybridization can detect gene mutations particularly deletions 
and insertions but also some base changes. For example the beta-globin gene 
probe detects an RFLP in MstI digested DNA which accurately predicts the 
occurrence of Sickle cell anaemia. The mutation destroys a restriction site,
thereby creating the polymorphism. Fetal DNA can be tested, as can parental 
DNA, and the occurrence of the disease prenatally diagnosed. The same probe
can detect deletions in beta-globin that cause thallasemias. Better probes 
for detecting base changes can be designed if the sequence of common and 
altered genes are known. An oligomer of 15-20 bp can be synthesized to be 
perfectly complementary to one type of gene and conditions for hybridization
found where only perfect matches hybridize. Both methods are useful where 
disease causing genes are known and their alterations relatively common in 
a population (egs. Sickle cell anaemia/malaria resistance, thallasemias, 
osteogenesis imperfecta, alpha antitrypsin deficiencies in humans, 
T-cytoplasmic male sterility in corn).

    	However genes of known function which work as specific probes to 
detect disease causing polymorphisms are rare. Often we need to sequence 
genes to detect differences single base pair differences OR infer a 
genotype by linkage to a gene/probe combination which shows an RFLP. More 
on both these techniques in the following lectures.

4.  Molecular Fingerprinting:

	Molecular fingerprinting is an extension of the RFLP technique 
which uses non specific probes, probes for genes where function is unknown. 
A probe is selected which detects many genes spread throughout the genome, 
minisatellite DNA, which contains lots of polymorphism. A hybridization 
probe made from the core sequence will simultaneously detect many bands on 
one blot, these bands will be specific to an individual. In the example 
mother and son share 25 of 61 bands, the chances of this at random are 
very low 2 in a zillion. Chances of an aunt posing as a mother are low, 6 
in a million. Probes of this type can be adapted for use with PCR and used 
in forensic evidence (blood, semen), paternity cases, patent protection 
variety improvement and variety protection in agricultural breeding 
programs. Though some modifications are made for plants due to the low 
occurence of the minisatellite sequences.  Markers such as RAPD, DAF, AFLP, 
and SAMPL are used to overcome this.