Hoàng Thị Mỹ Hạnh

Recombinant DNA Technology

Recombinant DNA technology procedures by which DNA from different species can be isolated, cut and spliced together -- new "recombinant " molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, yeast).

Application of Recombinant DNA

Human gene therapy: recombinant human insulin, recombinant human growth factor, recombinant hepatitis B vaccine…

Engineered crop plants: golden rice (β-carotene), herbicide resistant crops, insect resistant crops…

Recombinant DNA Tool

Vector

Enzyme used in molecular biology

Host cells

Restriction enzyme Recognize and cut at DNA specific sequence

DNA ligase Join compatible ends of DNA fragment. Use ATP

Alkaline phosphatase Remove phosphate group from strand of DNA

Polynucleotide kinase Add phosphate group to a DNA strand in the 5’ to 3’ direction

DNA polymeraseI DNA synthesize

Exonuclease III Digest nucleotide from a DNA strand in the 3’ to 5’ direction

RNAse Nuclease digest RNA, not DNA

Taq DNA polymerase Heat-stable DNA polymerase isolated from thermostable microbe.

Restriction enzymes are primarily found in bacteria and are given abbreviations based on genus and species of the bacteria.

One of the first restriction enzymes to be isolated was from EcoRI

EcoRI is so named because it was isolated from Escherichia coli strain called RY13.

Restriction Enzymes

Classify restriction enzymes

Type I enzymes are complex, multisubunit, combination restriction-and-

modification enzymes that cut DNA at random far from their recognition

sequences.

Type II enzymes cut DNA at defined positions close to or within their

recognition sequences.

Type III enzymes are also large combination restriction-and-modification

enzymes. They cleave outside of their recognition sequences and require two

such sequences in opposite orientations within the same DNA molecule to

accomplish cleavage; they rarely give complete digests.

Type IV enzymes recognize modified, typically methylated DNA and are

exemplified by the McrBC and Mrr systems of E. coli.

Restriction Enzymes

Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”.

Cut up “foreign” DNA that invades the cell.

Type II and III restriction enzymes cleave DNA chains at selected sites.

Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage.

An enzyme that recognizes a 6-base sequence is called a "six-base cutter”.

Type II restriction enzyme

EcoRI – Escherichia coli strain R, 1st enzyme

BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme

DpnI – Diplococcus pneumoniae, 1st enzyme

HindIII – Haemophilus influenzae, strain D, 3rd enzyme

BglII – Bacillus globigii, 2nd enzyme

PstI – Providencia stuartii 164, 1st enzyme

Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme

KpnI – Klebsiella pneumoniae, 1st enzyme

Basics of type II Restriction Enzymes

No ATP requirement.

Recognition sites in double stranded DNA have a 2-fold axis of symmetry – a “palindrome”.

Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends.

Results of Type II Digestion

Enzymes with staggered cuts complementary ends

HindIII - leaves 5´ overhangs (“sticky”)

5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’

3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’

KpnI leaves 3´ overhangs (“sticky”)

5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’

3’--CCATGG-- 5’ 3’ –C CATGG-- 5’

Results of Type II Digestion

Enzymes that cut at same position on both strands leave “blunt” ends

SmaI

Isochizomer XmarI

5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’

3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’

Compatible sticky ends

Restriction Endonucleases Cleave DNA at specific DNA sequences

Converting sticky ends to blunt ends

Filling in Trimming back

Converting blunt end to sticky end by using linker

Converting blunt end to sticky end by using linker

DNA Ligase in Action!

Electrophoresis

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

Gel: Agarose or Acrylamide

Dye

Nucleic acid stain: EtBr

Marker

Running time

Electrophoretic mobility of form of plasmid DNA

Circular DNA

Linear DNA

Supercoil DNA

Hypersupercoil DNA

Uncut plasmid DNA can be in five forms

Nicked

Circular

Linear covalently close

Supercoil

Hyper supercoil