Forensic DNA Fingerprinting: Using Restriction Enzymes

DNA FingerprintingProcedure Overview

DNA is Tightly Packaged into

Chromosomes Which Reside in the Nucleus

Model of DNA

DNA is Comprised of Four Base Pairs

DNA structure

DNA restriction enzymesEvolved by bacteria to protect against viral DNA infection

Endonucleases = cleave within DNA strands

Over 3,000 known enzymes

Enzyme Site Recognition • Each enzyme

digests (cuts) DNA at a specific sequence = restriction site

• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)

Palindrome

Restriction site

Fragment 1 Fragment 2

5 vs 3 Prime Overhang

• Generates 5 prime overhang

Enzyme cuts

Common Restriction Enzymes

EcoRI– Eschericha coli– 5 prime overhang

Pstl– Providencia stuartii– 3 prime overhang

The DNA DigestionReaction

Restriction Buffer provides optimal conditions

• NaCI provides the correct ionic strength

• Tris-HCI provides the proper pH

• Mg2+ is an enzyme co-factor

DNA Digestion

Temperature

Why incubate at 37°C?• Body temperature is optimal for these

and most other enzymes

What happens if the temperature is too hot or cool?

• Too hot = enzyme may be denatured (killed)

• Too cool = enzyme activity lowered, requiring longer digestion time

Restriction Fragment Length PolymorphismRFLP

Allele 1

Allele 2

GAATTCGTTAAC

CTGCAGGAGCTC

CGGCAGGCGCTC

PstI EcoRI

3Fragment 1+2Different Base PairsNo restriction site

M A-1 A-2

Electrophoresis of restriction fragments

M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments

AgaroseElectrophoresis

Loading

Electrical current carries negatively-charged DNA through gel towards positive (red) electrode

Power Supply

Buffer

Agarose gel

AgaroseElectrophoresis

Running

• Agarose gel sieves DNA fragments according to size

• Small fragments move farther than large fragments

Power Supply

Gel running

Analysis of Stained Gel

Determinerestriction fragmentsizes

• Create standard curve using DNA marker

• Measure distance traveled by restriction fragments

• Determine size of DNA fragments

Identify the relatedsamples

Molecular Weight

Determination

Size (bp) Distance (mm)

23,000 11.0 9,400 13.0

6,500 15.0

4,400 18.0

2,300 23.0

2,000 24.0 100

10,000

100,000

0 5 10 15 20 25 30

Distance, mm

Fingerprinting Standard Curve: Semi-log

ElectrophoresisEquipment

Forensic DNA Fingerprinting: Using Restriction Enzymes

Documents

Restriction Enzymes Aims: Must be able to recall the basic functions of restriction enzymes. Should be able to outline how specific restriction enzymes

Forensic DNA Fingerprinting: Using Restriction Enzymes

Restriction Endonucleases Or Restriction Enzymes

DNA Fingerprinting by Restriction Enzyme Patterns · DNA Fingerprinting by Restriction Enzyme Patterns ... whereby the genomic DNA of an organism is analyzed by ... DNA FINGERPRINTING

WHAT ARE RESTRICTION ENZYMES? Restriction Enzymes – molecular scissors able to cut DNA nuclease Enzymedigests nucleic acids

Enzymes Used for Gene Engineering 1. Restriction endonuclease (restriction enzymes) 2. DNA modifying enzymes a polymerases b nucleases c enzymes that modify

Restriction Enzymes. Restriction Endonucleases Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have

DNA Fingerprinting II - Austin Community College District · DNA Fingerprinting II Usage of Restriction Enzymes in DNA Fingerprinting Analysis EVT 002127K 3 EDVOTEK - The Biotechnology

AP Biology 2007-2008 DNA Fingerprinting AP Biology Many uses of restriction enzymes… Now that we can cut DNA with restriction enzymes… we can cut

Restriction Enzymes

Restriction enzymes treatment of DNA

Forensic DNA Fingerprinting: Using Restriction Enzymes STAM Winnipeg Oct 2013 Inspire your students – tomorrow’s researchers

DNA Fingerprinting: Gel Electrophoresis - Mrs. … Fingerprinting: Gel Electrophoresis Background Information: Restriction enzymes or restriction endonucleases are essential tools

FEATURES Restriction Enzymes

Cloning and Restriction Enzymes

Forensic DNA Fingerprinting Lab...Forensic Fingerprinting Lab Objectives •Use restriction enzymes to detect differences in the base sequences of individuals •Use gel electrophoresis

Restriction Enzymes. Theoretical Basis Using Restriction Enzymes The activity of restriction enzymes is dependent upon precise environmental condtions:

DNA Fingerprinting of Streptococcus pneumoniae Pulsed ... · enzymes throughout the entire chromosome, and DNA restriction fragment length polymorphisms of rRNAgenes (ribotyping)

DNA Fingerprinting Using Restriction Enzymes · DNA Fingerprinting Using Restriction Enzymes Background Information Background Information SOUTHERN BLOT ANALYSIS RFLP analysis of

Restriction Enzymes and Vectors