Restriction Enzymes and Gel Electrophoresis Genetic Engineering I Restriction Enzymes and Gel Electrophoresis

How are changes made to DNA Genetic engineering: Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules Different techniques are used to: Cut DNA into smaller pieces Identify the sequence of bases in a DNA molecule extract DNA from cells Make unlimited copies of DNA

The tools of molecular biology Cutting DNA: restriction enzymes are used to cut DNA into smaller pieces Separating different length of DNA: gel electrophoresis is used to separate different fragments of DNA by their size

Restriction Enzymes Naturally found in different types of bacteria: Bacteria use restriction enzymes to protect themselves from foreign DNA injected by viruses Function: Restriction enzymes cut DNA at specific sequences, they are also called “ molecular scissors”

Restriction Enzymes There are 2 types of bonds found in DNA: Covalent bonds : found inbetween nucleotides within a single strand Hydrogen bonds: found inbetween the 2 strands

Restriction Enzymes Restriction enzymes cut the covalent bonds of DNA molecules at specific sequences called restriction site:

Do you notice anything interesting about the sequence of the two strands? 5’ GAATTC 3’ 3’ CTTAAG 5’ Remember: DNA is composed of anti-parallel strands Palindrome sequence: a sequence that is the same whether read 5’ to 3’ on one strand or 5’ to 3’ on the complementary strand Some English palindromes: Dad, racecar, nurses run

Restriction Enzymes When restriction enzymes cut they produce either: Sticky ends: Blunt ends:

Gel electrophoresis Gel electrophoresis separates molecules on the basis of their charge and size: DNA molecules are negatively charged Different sized DNA fragments are placed in a gel made of agarose (a sugar found in seaweed) Electricity is applied to the gel DNA fragments will move across the gel at a speed dependent on their size: the longer DNA fragments will move slower the shorter DNA fragments will move faster

Gel electrophoresis Wells

Gel electrophoresis

Applications for restriction enzymes and gel electrophoresis 1. Evolutionary study: Comparing DNA samples from different organisms to measure evolutionary relationships turtle snake rat squirrel fruitfly – 1 3 2 4 5 1 2 3 4 5 DNA  +

Applications for restriction enzymes and gel electrophoresis 2. Medical diagnostic: Comparing normal alleles to disease alleles: e.g. test for Huntington's disease allele 1 allele 2 – DNA  chromosome with normal allele 1 chromosome with disease-causing allele 2 +

Application for restriction enzyme and gel electrophoresis 3. Forensics: comparing DNA samples from a crime scene with suspects & victim suspects crime scene sample S1 S2 S3 V – DNA  +

Application for restriction enzyme and gel electrophoresis 4. Paternity: Who’s the father? – Mom F1 F2 child DNA  +