Restriction enzymes Flashcards
20.1 Recombinant DNA Technology Began with Two Key Tools:
Restriction Enzymes and Cloning Vectors
Recombinant DNA technology is aka
Genetic Engineering
Recombinant DNA technology/ genetic engineering is based on the ______________ of DNA molecules at __________________.
Resulting DNA fragment can be transferred from any organism to a virus or bacterial cell in order to ____________
cutting and pasting
specific base sequences
mass-produce, isolate and characterise it.
Recombinant DNA technology/ genetic engineering is used for
gene mapping
diagnosis of diseases
commercial production of human gene products
expression of foreign genes in transgenic animals and plants.
Making recombinant DNA
The process comprises a series of steps:
- DNA is isolated from cells
- DNA is cut by restriction enzymes (REs)
- Fragments are ligated into “vectors”
- Recombinant molecules are transformed into host cells
- Multiplication occurs in host cells
- Cloned DNA can be recovered, purified and analysed
Function of restriction Enzymes
Cut DNA at Specific Recognition sequences
How are Restriction Enzymes produced?
Produced by bacteria as a means to combat bacteriophage infection.
= Degrade the DNA of infecting viral pathogens.
[Endonucleases (antiviral protection against phages)]
How do Restriction Enzymes cut DNA sequences?
Cuts both strands of DNA within the recognition sequence by cleavage of the phosphodiester backbone.
Some common restriction enzymes, outlining their (palindromic) recognition sequences. Note their diverse DNA cutting patterns, and source microbes. The arrows indicate the site in the DNA where each respective enzyme will cut.
Characteristics of restriction enzymes
- Two types: Type I and II
- > 500 commercial REs
- Palindromic recognition sequences
- Generate restriction fragments
- Frequent vs. rare cutters
- Sticky (cohesive) vs. blunt ends
Recognition sequences of restriction enzymes occur randomly in the genome as palindromes, what does this mean?
they read the same on both strands of DNA.
Read in a 5’-3’ and a 3’-5’ orientation
Recognition sequences of restriction enzymes are named after the
organism from which they are discovered
EcoRI was the first restriction enzyme discovered from
Escherischia coli strain R.
Depending on their recognition sequences, restriction enzymes can produce either
sticky” or “blunt” ends
Sticky ends
Sticky ends create overhangs
Blunt ends
create blunt ends
Pieces of DNA which have sticky ends generated by the same restriction enzyme are able to
anneal because of complementary base pairing.
Pieces of DNA which have sticky ends generated by the same restriction enzyme are able to anneal because of complementary base pairing.
But there are still nicks left after this.
DNA ligase will seal the nick via ligation
Explain ligation
DNA ligase will seal the nick in the phosphodiester backbone of DNA by using ATP to catalyze the covalent joining of the two fragments together, forming recombinant DNA molecules.
Restriction enzyme EcoRI comes from
Escherichia coli R
Restriction enzyme HindIII comes from
Haemophilus influenzae RD
Restriction enzyme BamHI comes from
Bacillus amyloliquefaciens H
Restriction enzyme TaqI comes from
Thermus aquaticus
- What is the palindromic recognition sequence of HindIII?
- What does the III stand for in HindIII?
- Does HindIII generate a blunt end or sticky end??
- AAGCTT
- It is the third restriction enzyme discovered from H. influenzae Rd
- Sticky ends
Which of the following is a palindromic recognition sequence?
A)5’. . . CAATAG . . . 3’
B) 5’. . . CAATTG . . . 3’
C) 5’. . . CATTTG . . . 3’
D) All of the above
E) None of the above
B
CAATTG
GTTAAC
