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Restriction Enzymes and Gel Electrophoresis

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

2 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

3 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

4 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”

5 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

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

7 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

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

9 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

10 Gel electrophoresis Wells

11 Gel electrophoresis

12 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 +

13 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 +

14 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 +

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

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