DNA Fingerprinting11.06.2008

Catalyst:

What are polymorphisms?

What is “junk” DNA? How do you think it is used?

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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.

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.

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DNA Identification

Four main steps of DNA Fingerprinting:

1. Extraction (we did this during Friday’s lab – will not cover again today)

2. Restriction Fragments3. Amplification4. Electrophoresis

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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.

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VNTR / STR

VNTR and STR data are analyzed for (a) tissue matching and (b) inheritance matching.

Restriction Fragments Step 2:

The DNA is cut into fragments using restriction enzymes.

Each restriction enzyme cuts DNA at a specific base sequence.

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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.

Amplification / PCRPurpose – 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 / PCRTemperature 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 temperatureOnline Demo: http://www.dnalc.org/ddnalc/resources/pcr.html

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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.

ElectrophoresisStage 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.

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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.

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