DNA Fingerprinting and the Polymerase Chain Reaction

Sequence Repeats in the Human Genome

Variable Number of Tandem Repeats (VNTR): repeats of 9 to 80 base pairs (bp), total length is 500 to 23,000 bp, very specific due to length and repeats, testing is expensive and time-consuming, degrade in older DNA samples due to random breaking of DNA strands Amplified Fragment Length Polymorphisms (AmpFLP): repeats of 8 to 16 bp, total length 100 to 1300 bp, shorter and less susceptible to degradation, first loci to be used in forensic analysis Short Tandem Repeats repeats of 2 to 7 bases, total length 100 to 400 bp, shorter yet thereby less susceptible to breakage, these loci are the current standard in forensic laboratory analysis, ideal size for PCR amplification Single Nucleotide Polymorphisms (SNP): a single base change as a result of mutation, not commonly useful to forensic investigators, can be potentially used to distinguish identical twins

Polymerase Chain Reaction (PCR)

Technique devised in 1983 to amplify small amounts of DNA Can be performed on DNA from a single cell

- cigarette butt, a licked stamp, root of a single hair, 1/50,000 a drop of blood (0.1 microliters)
The amplified DNA can then be used to:

- identify a suspect or victim

-determine an individuals sex -determine species (if not human)

PCR to Amplify a Persons DNA

Steps Involved: 1. Isolate repeating loci from a persons DNA using restriction enzymes 2. Design primers short segments of synthetic DNA that are complementary to DNA on either side of the VNTR regions

3. Add vast excess of the primers and heat mixture to 75 oC This causes DNA strands to separate by breaking hydrogen bonds between bases

4. Cool to 15 oC. Primers hydrogen bond (anneal) to complementary strands

5. Add DNA polymerase and all four types of nucleotides. The polymerase (enzyme used in DNA replication) will fill in the rest of the two strands.

You now have two identical copies of the DNA you started with.

6. Repeat steps. Heat to break hydrogen bonds. Cool to anneal more primers (still there in vast excess). Allow DNA polymerase to fill in the remaining strands. Two strands of DNA become four. EtcEtcEtc..

PCR
Originally, the DNA polymerase would have to be added between each heating step because it would fall apart at 75 degrees. Now, an enzyme called Taq DNA polymerase is added. This is a very stable enzyme isolated from bacteria living at thermal vents in the ocean (up to 95 oC) In just 32 rounds of PCR, 1 copy of DNA becomes 4.2 billion copies. This would take about 3 hours to perform in lab.

Pros and Cons???????

DNA Fingerprinting
Used to identify individuals by their RFLP (usually STR) regions

Steps involved: 1. Isolate and amplify DNA if needed 2. DNA is cleaved into smaller pieces with restriction enzymes DNA is separated with gel electrophoresis

3.

4. DNA is transferred to a nylon membrane (Southern blotting)

5. A radioactive primer is designed that will be complementary to unique regions (STR, etc, regions). Add this to nylon membrane containing DNA. 6. Wash off excess primer and hold nylon up to a photographic plate to expose. The pattern will be unique to the individual.

Clearly, suspect one is the match..

If all STR regions are considered, there is a one in 3.4 billion chance of error. This means there may be one other person on the planet that would be too similar to tell the difference.

If other RFLP regions are also considered, the chances of error go way, way down

Mitochondrial DNA
genetic material from the mitochondria (cellular organelle where energy is produced) inherited from the mother only

Advantages:
more sensitive (less DNA needed), degrades slower than nuclear DNA

can be used in cases where nuclear DNA cannot (hair without root, skeletal remains)
Disadvantages:

all people of same maternal line will be indistinguishable (less discriminatory)

more work, more time consuming, more costly

CODIS Combined DNA Index System

National software developed by the FBI Distributed to local, state, and national crime labs

All 50 states mandate inclusion of DNA fingerprint (if available) from violent and sexually motivated crimes
Mostly a database of STR regions

Thousands of matches have led to the capture of criminals that otherwise would not have been caught

This has led numerous people to suggest a national DNA database that would include only polymorphism information