TECHNIQUES IN CONSERVATION GENETICS

DNA FINGERPRINTING

By : Holly MacLeod

TABLE OF CONTENTS

PROCEDURE

ANALYSES

USES IN CONSERVATION BIOLOGY

DNA fingerprinting involves the use sequences of DNA which consist of several hundred 15-60 base pair units, repeated over and over. The purpose of the technique is to provide a way of measuring differences in the genetic makeup of different plants or animals within or between populations.

PROCEDURE

  1. Use clean high molecular weight DNA, usually extracted from blood.

  2. Digest about 5 ug of each sample with restriction endonucleases (most commonly used are HinfI, HaeIII, MboI, and AluI). These enzymes cut frequently so most DNA will be reduced to small fragments. Any large fragments remaining are likely repetitive sequences that are used by the DNA fingerprinting procedure.
  3. Perform electrophoresis on an agarose gel until the fragments have migrated the appropriate distances. This separates the fragments based on size.
  4. Correlate running times with times taken by fragments of known sizes.
  5. Transfer the DNA to a membrane which allows the fragments to be held down for hybridization.
  6. Radioactive probes (a known sequence of about 12-60 base pairs in length) are added to the membrane and allowed to stick to any pieces of the DNA to which they are complementary.
  7. Since the probes are radioactive, they will 'light up' the DNA sequences to which they have hybridized.
  8. The membrane is then washed to remove any of the pieces of DNA that have not stuck to probes.
  9. The remaining DNA on the membrane is exposed to X-ray film to obtain an image like that seen below.

IMAGE OF DNA FINGERPRINT

The final product of the DNA fingerprinting procedure is a pattern of bands, often specific to the individual (about 10-25% of the bands are shared between any 2 individuals by chance) except in extreme cases of inbreeding or in identical twins. The technique is used most often in studies to identify relationships between individuals, or to determine paternity in studies of reproductive behaviour and mating success. Comparison of populations can be done when the population size is sufficiently small to result in a reduction of individual variability. Under this sort of situation, population-specific banding patterns may be identified.

ANALYSES

Measure of homozygosity of the individual and the population:

Count the average number of bands in the fingerprint profiles. A low number of bands indicates a high proportion of homozygous alleles in the individual or population. Since homozygosity is related to the level of inbreeding, this is a useful test for conservation biology.

Similarity Index:

To calculate similarity between two individuals, the number of bands that are shared by the two must be counted. This number is multiplied by two and divided by the total number of bands seen for both individuals. DNA fingerprint profile similarity increases with increasing degree of relatedness.

Population Subdivision:

Measures of the genetic diversity extracted from single-locus analysis are used frequently to estimate measures of population subdivision. This is tested through use of the similarity index. It involves a statistical test to determine if there is a significant difference in the genetic makeups of individuals from two different populations.

HOW IS THIS TECHNIQUE USED IN CONSERVATION BIOLOGY

Relationships between individuals
This is very important in determining whether a population has lost a great deal of its genetic diversity as a consequence of having few individuals. It is also extremely useful in captive breeding programs. By identifying the relationships between all individuals in the captive population, the zoo-keepers can ensure that only unrelated individuals mate.

Level of population and individuals homozygosity
A high level of homozygosity in the group could indicate that inbreeding is occurring. This will threaten the fitness of the population and shows that this group should be closely monitored and could be of conservation concern.

Degree of population subdivision
If the similarity index between populations shows that there is very little genetic similarity between the two groups, this suggests that there is very little gene flow occurring by way of migration. Therefore, the two groups should be considered separately in conservation studies.

Wildlife forensics
Since DNA fingerprints are unique to the individual, they can be used to determine whether the carcass that a hunter has recently brought home is actually the same one that left the gut pile and blood in an area in which hunting is illegal.

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