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:1. Covalent bonds : found inbetween

nucleotides within a single strand2. Hydrogen bonds: found inbetween the 2

strands

Restriction Enzymes• Restriction enzymes cut the covalent bonds of

DNA molecules at specific sequences called restriction site:

• 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

Do you notice anything interesting about the sequence of the two strands?

• When restriction enzymes cut they produce either:

- Sticky ends:

- Blunt ends:

Restriction Enzymes

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 slowerthe shorter DNA fragments will move faster

Gel electrophoresisWells

Gel electrophoresis

Applications for restriction enzymes and gel electrophoresis

1. Evolutionary study: Comparing DNA samples from different organisms to measure evolutionary relationships

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DNA

1 32 4 5 1 2 3 4 5

turtle snake rat squirrel fruitfly

2. Medical diagnostic: Comparing normal alleles to disease alleles:e.g. test for Huntington's disease

chromosome with disease-causing allele 2

chromosomewith normal allele 1

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allele 1allele 2

DNA

Applications for restriction enzymes and gel electrophoresis

3. Forensics: comparing DNA samples from a crime scene with suspects & victim

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S1

DNA

S2 S3 Vsuspects crime

scene sample

Application for restriction enzyme and gel electrophoresis

4. Paternity: Who’s the father?

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DNA

childMom F1 F2–

Application for restriction enzyme and gel electrophoresis