Genetics 244
Chapter 20: Recombinant DNA Technology
Lecture 1:
- Also called genetic engineering
- Based on cutting and pasting DNA molecules at
specific base sequences
- The resulting fragment can be transferred from any
organism or virus into a bacterial cell
- The DNA fragment can be mass-produced, isolated
and characterized in the bacterial cell
Uses:
- Gene mapping
- Diagnosis of disease
- Commercial production of human gene products
- Expression of foreign genes in transgenic animals
and plants
- The first commercial “biotech product” was insulin
in 1982
Making recombinant DNA:
- The process is made up of several steps
- These steps are universally used for all
applications
1. DNA is isolated from cells
o Find a way to isolate the specific sequence you
are interested in
2. DNA is cut by restriction enzymes (REs)
3. DNA fragments are ligated (pasted) into vectors
, o Using ligases which fix phosphodiester bonds
4. Recombinant molecules are transformed into host
cells
5. Multiplication occurs in the host cells
6. Cloned DNA can be recovered, purified and
analyzed
Restriction enzymes:
- “Molecular scissors”
- A tool used in recombinant technology
- There is a defense mechanism of bacteria against
invading phages
- Endonucleases (REs) cut and inactivate the DNA of
the phage, allowing the phage to invade the
bacterial host cell
- Type II REs recognize and cut at the exact place
- Palindromic recognition sequences (3’ and 5’ are
the same sequence backwards)
- Generate restriction fragments
Frequent vs rare cutters
- Frequent cutters recognize six-base sequences
- 46= 4096bp
- Rare cutters recognize four-base sequences
, - 44= 356bp
- Both frequent and rare cutters always recognize
palindromic sequences
Sticky vs blunt ends
- Sticky (cohesive) ends cut with overhang
- Recognize 3’ vs 5’ overhangs
- Overhangs are complementary and can be fixed by
ligase
- Blunt ends do not have an overhang
- They are difficult to ligate because there are no
complementary base pairs to fix
Creating a recombinant DNA molecule
Chapter 20: Recombinant DNA Technology
Lecture 1:
- Also called genetic engineering
- Based on cutting and pasting DNA molecules at
specific base sequences
- The resulting fragment can be transferred from any
organism or virus into a bacterial cell
- The DNA fragment can be mass-produced, isolated
and characterized in the bacterial cell
Uses:
- Gene mapping
- Diagnosis of disease
- Commercial production of human gene products
- Expression of foreign genes in transgenic animals
and plants
- The first commercial “biotech product” was insulin
in 1982
Making recombinant DNA:
- The process is made up of several steps
- These steps are universally used for all
applications
1. DNA is isolated from cells
o Find a way to isolate the specific sequence you
are interested in
2. DNA is cut by restriction enzymes (REs)
3. DNA fragments are ligated (pasted) into vectors
, o Using ligases which fix phosphodiester bonds
4. Recombinant molecules are transformed into host
cells
5. Multiplication occurs in the host cells
6. Cloned DNA can be recovered, purified and
analyzed
Restriction enzymes:
- “Molecular scissors”
- A tool used in recombinant technology
- There is a defense mechanism of bacteria against
invading phages
- Endonucleases (REs) cut and inactivate the DNA of
the phage, allowing the phage to invade the
bacterial host cell
- Type II REs recognize and cut at the exact place
- Palindromic recognition sequences (3’ and 5’ are
the same sequence backwards)
- Generate restriction fragments
Frequent vs rare cutters
- Frequent cutters recognize six-base sequences
- 46= 4096bp
- Rare cutters recognize four-base sequences
, - 44= 356bp
- Both frequent and rare cutters always recognize
palindromic sequences
Sticky vs blunt ends
- Sticky (cohesive) ends cut with overhang
- Recognize 3’ vs 5’ overhangs
- Overhangs are complementary and can be fixed by
ligase
- Blunt ends do not have an overhang
- They are difficult to ligate because there are no
complementary base pairs to fix
Creating a recombinant DNA molecule
