DNA Fingerprinting Flashcards
Restriction Fragment Length Polymorphism (RFLP)
A technique used to distinguish variations in homologous DNA sequences
Restriction Enzymes (Endonucleases)
Enzymes that cut DNA at specific sequences (recognition sites)
Palindrome Sequence
A sequence of DNA that reads the same forward and backward (e.g., GAATTC for EcoRI)
Agarose Gel Electrophoresis
A method used to separate DNA fragments based on size by applying an electric current.
Micropipette
A precision instrument used to measure and transfer small volumes of liquid
DNA Ladder
A molecular weight marker used to estimate the sizes of DNA fragments in gel electrophoresis
RFLPs are generated by?
cutting DNA with restriction enzymes, producing fragments of different lengths.
The fragments are separated using
gel electrophoresis
reveals the banding patterns
RFLP analysis helps
forensic identification, paternity testing, and genetic mapping.
Restriction Enzymes
Named after the bacterial species they are derived from (e.g., EcoRI comes from Escherichia coli strain RY13).
What do Restriction Enzymes do?
They recognize specific sequences (usually palindromic) and cut DNA at those sites.
What are Restriction Enzymes used for?
Used in genetic engineering, forensic analysis, and molecular cloning.
How Restriction Enzymes Work
Scan DNA sequences and locate their specific recognition site.
Cut the DNA at or near this site, creating blunt or sticky ends.
Example: EcoRI recognizes GAATTC and cuts between G/AATTC.
EcoRI Palindrome Sequence
GAATTC (5’ → 3’)
CTTAAG (3’ → 5’)
Cut occurs between G/AATTC producing sticky ends.
Restriction Enzymes Used in Lab
Common restriction enzymes: EcoRI, HindIII, BamHI.
Reagents Used in Lab
Loading dye: Helps track DNA migration in the gel.
Agarose gel: Provides a medium for DNA separation.
Buffer solution: Maintains pH and ionic strength for enzyme activity.
Agarose Gel Electrophoresis Setup
Prepare and pour the agarose gel into a casting tray.
Load DNA samples into wells using a micropipette.
Submerge the gel in buffer and apply an electric current.
DNA (negatively charged) moves toward the anode (+).
Smaller fragments travel farther, while larger fragments remain closer to the wells.
Agarose Gel Electrophoresis theory
DNA fragments are separated based on size (smaller fragments move faster and farther).
Reliability of DNA Evidence and DNA Fingerprinting
Highly reliable if proper procedures are followed.
No two individuals (except identical twins) have identical DNA patterns.
Used in criminal investigations, paternity tests, and identifying remains.
Errors can occur due to contamination, mislabeling, or improper handling.
Proper Micropipette Usage
Choose the correct pipette (P10, P100, P1000) based on volume.
Set the volume correctly.
Use the correct pipetting technique:
Press to the first stop to draw liquid.
Release slowly to avoid bubbles.
Dispense by pressing to the second stop.
Change tips between samples to prevent contamination.
Reading a Gel and Banding Patterns
Interpretation:
Compare banding patterns between the crime scene DNA and suspects.
The suspect whose banding pattern matches the crime scene sample is likely the source of the DNA.
Restriction Sites:
The more restriction sites an enzyme cuts, the more DNA fragments appear on the gel.
Diagram of Agarose Gel and Banding Patterns
Typical Gel Layout:
Lane 1: DNA Ladder (size marker)
Lane 2: Crime Scene Sample
Lanes 3-6: Suspect DNA Samples
Bands at the same positions indicate a match.
