DNA Fingerprinting Catalyst: What are polymorphisms? 11.06.2008 Catalyst: What are polymorphisms? What is “junk” DNA? How do you think it is used?

DNA Identification Junk DNA contains many of the unique patterns of repeated base sequences that identify individuals. In a human population, these are called polymorphisms. In 1984 a technique was developed for isolating and analyzing these variable areas. This DNA Fingerprinting appears as a pattern of bands on X-ray film. These patterns can be used for identification of individuals. Forensic Science: Fundamentals & Investigations, Chapter 7

Who Invented it? The process of DNA fingerprinting was invented by Alec Jeffreys at the University of Leicester in 1985. He was knighted in 1994.

Famous Cases Colin Pitchfork (yes, this is his name) was the first criminal caught based on DNA fingerprinting evidence. He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.

DNA Identification Four main steps of DNA Fingerprinting: Extraction (we did this during Friday’s lab – will not cover again today) Restriction Fragments Amplification Electrophoresis Forensic Science: Fundamentals & Investigations, Chapter 7

Variable Number of Tandem Repeats (VNTR) VNTR / STR The number of copies of the same repeated base sequence in DNA varies among individuals. Variable Number of Tandem Repeats (VNTR) Within junk DNA, sequences of DNA are repeated multiple times. Some can be 9-80 bases in length. Short Tandem Repeats (STR) Within junk DNA, other sequences of DNA also are repeated multiple times. These usually are only 2-5 bases in length and are becoming the preferred sequences for analysis. Forensic Science: Fundamentals & Investigations, Chapter 7

VNTR / STR VNTR and STR data are analyzed for (a) tissue matching and (b) inheritance matching. Forensic Science: Fundamentals & Investigations, Chapter 7

Restriction Fragments Step 2: The DNA is cut into fragments using restriction enzymes. Each restriction enzyme cuts DNA at a specific base sequence.

Amplification With some VNTR analysis, polymerase chain reaction (PCR) can be used to amplify the DNA that contains the VNTRs. In STR profiles, restriction enzymes are unnecessary; PCR allows the amplification of the strands with STR sequences. Forensic Science: Fundamentals & Investigations, Chapter 7

Amplification / PCR Purpose – Quickly make many copies of a region of a DNA molecule Method – Multiple rounds of DNA replication Components in PCR reaction – Target DNA, nucleotides, DNA polymerase, and primers Temperature cycling – DNA replication controlled by temperature…

Amplification / PCR Temperature cycling – PCR process uses a machine (thermocycler) in which PCR reaction goes through ~30 cycles of three different temperature changes: ~95ºC – Melting temperature 50-65ºC – Annealing temperature 72ºC – Extension temperature Online Demo: http://www.dnalc.org/ddnalc/resources/pcr.html

Electrophoresis DNA samples are placed in gels through which electronic currents are passed. DNA fragments line up in bands along the length of each gel. Forensic Science: Fundamentals & Investigations, Chapter 7

Electrophoresis Stage 4: Fragments are separated on the basis of size using a process called gel electrophoresis. DNA fragments are injected into wells and an electric current is applied along the gel.

Electrophoresis DNA is negatively charged so it is attracted to the positive end of the gel. The shorter DNA fragments move faster than the longer fragments. DNA is separated on basis of size.

Electrophoresis A radioactive material is added which combines with the DNA fragments to produce a fluorescent image. A photographic copy of the DNA bands is obtained.

In most criminal cases, 6-8 probes are used. DNA probes are used to identify the unique sequences in a person’s DNA. Different DNA probes are made up of different synthetic sequences of DNA bases compli-mentary to the DNA strand. The probe binds to complimentary bases in the strand (see the fragmentary DNA bands above). In most criminal cases, 6-8 probes are used. Forensic Science: Fundamentals & Investigations, Chapter 7

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…

Foldable & Comparison to DNA Replication