History of DNA Fingerprinting

In 1984, Dr. Alec Jeffreys developed a technique for isolating and analyzing sequences of DNAHe called this procedure DNA

FingerprintingIn 1985, Dr. Kary Mullis invented

the PCR technique allowing for the creation of a DNA profile from trace amounts of DNA

Human Genome

The genome is the total amount of DNA in the nucleus of an organismHumans have about 3 million base pairs of DNAMost of the human genome is the same from

one person to another, but there are variationsThe variations, which occur in the non-coding

regions of the DNA, consist of unique patterns of end to end repeated base sequences called tandem repeats

Tandem Repeats

The number and location of the tandem repeats are unique in each individual so they create a unique DNA profileThese repeats may be studied to aid in the identification of individualsThe more locations in an individual’s DNA that are examined, the higher the probability that you can identify the individual

Variable Number of Tandem Repeats (VNTRs)

The number of copies of the same repeated base sequence in the DNA can vary among individualsEx: the sequence ACTGACGATC might be

repeated 3 times in one person, but 7 times in another personVNTRs can be 9 to 80 bases long

Short Tandem Repeat (STR)

Short sequence of DNA, usually only two to five base pairs in length, within the non-coding DNASTRs are the preferred method of analysis

because they are more accurate and can be used with small or partially degraded samples of DNAVNTRs are longer and require the DNA to be

longer, making it difficult to separate the VNTR sequences

Uses for a DNA Profile

Tissue Matching: used to match crime scene evidence to a suspect; the two samples must have the same band patternInheritance Matching:

each band in a child’s DNA fingerprint must be present in at least one parent

DNA Directionality

One of the parent strands in DNA runs in a 5’ to 3’ direction while the other runs in a 3’ to 5’directionThe 3 and 5 refer to the

carbon number of the deoxyribose ring

Primers and Polymerase

DNA Primers are short segments of DNA that are complementary to the target DNADNA Polymerase is the enzyme that binds free-floating nucleotides to the complementary bases on a DNA strandRestriction Enzymes are proteins that recognize a particular sequence in DNA and cut the DNA apart at that location

Steps in DNA Fingerprinting

1. Extraction2. Restriction Fragments3. Amplification4. Electrophoresis

Extraction

DNA must be removed from the nucleus of the cells

Restriction Fragments

Restriction enzymes are used to cut apart the DNA at specific sites

Amplification

Polymerase Chain Reaction (PCR) generates multiple identical copies from trace amounts of original DNA evidenceEnable forensic scientists to make billions of

DNA copies from small amounts of DNA in just a few hours

Steps in PCR

1. Mix the primers with DNA, DNA Polymerase, buffer and nucleotides

2. Heat the mixture to boiling to denature the DNA

Steps in PCR (cont.)

3. Allow the mixture to cool4. At this point, the DNA would normally re-zip

to form the original double-stranded molecule, but the primers attach to the DNA instead

Steps in PCR (cont.)

5. DNA polymerase will now bind nucleotides to the end of each primer to complete the complementary strands

6. There are now 2 complete copies of the DNA7. The entire process is repeated over and over

again to create millions of fragments of DNA

Electrophoresis

In this process, DNA fragments created through PCR are separated by using an electrical fieldDNA is negatively charged and will move

towards a positive electrodeThe smaller the fragment, the faster it will

travel

Steps in Electrophoresis

1. Preparing the Buffer: add 25 ml of 20x TBE to 475 ml of distilled water

2. Preparing the Gel: melt the agarose and let it cool

3. Pour the Gel: place a comb into the gel box and pour the gel into the box so that it flows between the teeth of the comb; do not spill the gel into the areas at either end of the box; let the gel set

Steps in Electrophoresis

4. Load the Gel: pour TBE solution into the gel box so that it covers the surface of the gel and floods the areas at the ends of the box; pull the comb out; using a pipette draw up the DNA and dye out of the tubes and load into the well

Steps in Electrophoresis

5. Adding electrodes: use carbon filter paper as electrodes; use alligator clips to attach the electrodes to the power supply

Steps in Electrophoresis

6. Running the Gel: turn the power on and let the gel “run”; do not disturb the box while the gel is running; once the blue dye reaches the end of the gel, turn off the power

7. Staining the DNA: pour blue staining solution on top of the gel and let it sit 4 minutes; rinse the gel 3-4 times and leave the gel overnight to develop

8. Destaining: destain the gel again; leave water in the box and change it about 4 times to gradually wash away the “background” stain

Southern Blotting

The DNA on the gel can now be transferred to a nylon membrane in a procedure called Southern BlottingThe bands of DNA on the membrane are the

DNA fingerprintA radioactive piece of DNA called a probe is

used to locate complementary sequences on the membrane

CODIS

Combined DNA Index System: an electronic database of DNA profilesIndividuals who have been convicted of

certain crimes (i.e. rape, murder, child abuse) have their DNA profiles entered into the database