ACTIVITY 2. SEPARATING THE DNA FRAGMENTS


Activities:
INTRODUCTION
ACTIVITY 1. TARGETING THE DNA FOR DNA FINGERPRINTING
Repeated sequences of non-coding DNA
CODIS: the forensic database
ACTIVITY 2. SEPARATING THE DNA FRAGMENTS
Agarose gel electrophoresis
Capillary electrophoresis
ACTIVITY 3. OBTAINING ALLELIC DNA FRAGMENTS
The RFLP method of DNA fingerprinting
The PCR method of DNA fingerprinting
ACTIVITY 4. TESTING YOUR UNDERSTANDING


ACTIVITY 2. SEPARATING THE DNA FRAGMENTS

Agarose gel electrophoresis

Any separation of DNA fragments containing allelic regions to be compared among individuals is done by electrophoresis. The form of electrophoresis that is used in most research laboratories to separate pieces of DNA is called slab or agarose-gel electrophoresis.In this method, the DNA fragments are separated according to size. Thus, the smallest DNA fragments move the fastest and are found at the bottom of the gel when the run is finished. Since DNA is negatively charged, the fragments move down the gel toward the positive pole. View the following videos which show the preparation of the agarose gel and assembly of the electrophoretic apparatus.

video — Preparing the agarose gel

video — Assembling the electrophoretic apparatus

Most introductory laboratories that demonstrate DNA electrophoresis utilize very short DNA segments of different lengths that can be separated in an hour or so. Below is a photograph of a simulated crime scene based on analysis of such fragments. The 6 lanes of DNA fragments exhibit banding patterns that can be used comparatively, in this case to match evidence with a suspect.

classroom gel

Separation of DNA fragments on an agarose gel

Lane 1 contains the victim’s DNA fragments, lane 2 the DNA evidence, and lanes 3-6 DNA from four suspects.


In the Genetic Technology & Bioinformatics topic, you have seen a real DNA fingerprint that was used several years ago as evidence in a trial. This older method of DNA fingerprinting was based on the banding patterns of DNA fragments produced by agarose gel electrophoresis. Note that several different fingerprints representing comparisons of several loci were always used to exclude (or include) a potential suspect when this method was used. View the following animation to summarize the agarose gel method.

animation – DNA electrophoresis

When you understand how agarose gel electrophoresis works and how it has been used in DNA fingerprinting, answer questions 2 and 3.

Capillary electrophoresis

Separation of DNA fragments by agarose gel electrophoresis is still a very powerful tool in the research laboratory (which is why it is featured in this laboratory assignment). It is still used to help diagnose and follow the inheritance of some diseases. However, in forensic science it has been replaced by capillary gel electrophoresis. This type of electrophoresis uses narrow silica capillaries (tubes) containing a polymer solution through which the negatively charged DNA molecules migrate under the influence of a high voltage electric field. It is fast, easily automated, and allows analysis of smaller amounts of DNA than required for agarose gel electrophoresis.

capillary electrophoresis This diagram shows the basic setup of a capillary electrophoresis system. Note that a computer is attached to a detector at the bottom of the capillary tube. For more information on the system, view these photographs.

Whereas agarose gels produce a banding pattern of DNA fragments, the capillary method is computerized and produces a pattern of peaks on a graph.

DNA graph

In the capillary method, the DNA fragments are labeled to make them fluorescent. A detector measures fluorescence as the fragments pass through the capillary tube. Each peak on the graph represents a fragment of different molecular weight (size). The pattern of peaks is the DNA fingerprint of one individual. Thus, it corresponds to one lane of bands in an agarose gel.

When your understand how the capillary method differs from gel electrophoresis, answer question 4.

ACTIVITY 3. OBTAINING ALLELIC DNA FRAGMENTS

Be sure to review the material on restriction enzymes before beginning this activity.

The RFLP method of DNA fingerprinting

There are two ways to obtain the DNA fragments needed for fingerprinting, and sometimes they are combined in specific applications. Jeffreys subjected nuclear DNA to restriction enzyme digestion. The fragments obtained are known as RFLPs  because they represent restriction fragments with length polymorphisms. The DNA sequences targeted were arrays of moderately repetitive and short (10-60 bases) DNA sequences which are dispersed throughout the genome. They are known as “variable number tandem repeats” (VNTRs), and their degree of repetition is two to several hundred at each locus. There are thousands of loci, but each locus shows a distinctive repeat unit. For any one locus, most of the polymorphism observed among individuals is caused by variation in the number of repeats, although individuals can also vary in the number of sites to which a particular restrictive enzyme can attach while digesting the DNA into fragments. View the following animation which shows the digestion of DNA into fragments that vary in the number of repeats they contain.

animation – digestion of DNA into fragments

The resulting fragments are separated by size using agarose gel electrophoresis. The desired result is a banding pattern that reflects the differences in repeats that vary among individuals. However, the gel must be processed further to obtain a distinct banding pattern of allelic sites. This is because there are so many different restriction fragments on the gel, it usually appears as a smear rather than discrete bands. To obtain a discrete banding pattern, the DNA is denatured into single strands by incubation with NaOH, then transferred to a stable surface which is a sheet of nitrocellulose or nylon. This procedure is called “blotting”; the DNA fragments retain the same pattern of separation that they had on the gel. The blot is then incubated with probes that recognize specific allelic fragments.

probe Each probe is a single-stranded DNA that is complementary to the sequence of nucleotides in one or more of the restriction fragments. The probes are either radioactive or fluorescent to allow them to be detected by exposure to X-ray film.

 

rflp

This diagram illustrates the steps in DNA fingerprinting by the RFLP method. You should also view the animation below, since it includes some important details.

animation – the RFLP method of DNA fingerprinting

Criteria for identifying suspects are based on the probabilities derived from analyzing several gels, using probes for at least 5-10 different loci. Study the following example in which two loci are examined:

separation

Here are repetitive sites at two loci (A and B) which vary among individuals. This example shows the pattern for A and B in two individuals (sample 1 and sample 2).

sharing alleles

In this gel, the banding pattern of the suspect is the same as found in the evidence.

The frequency of each allele type in the population is known (left column).

The probability that any individual would share the same banding pattern with the evidence is 1 in 7,452,000 (product of frequencies for the 4 alleles). Thus, it appears that the suspect is guilty. Note that if more than 4 loci were examined, the probability of 2 individuals sharing the same alleles would be in the billions.

When you understand the RFLP method and how probabilities are calculated, answer questions 5 -8. For question 8, use this DNA fingerprint.

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