Recombinant DNA technology

Bionanotechnology 2013

 Recombinant DNA technology is the core capability of bionanotechnology. This technology allows to construct any protein that we wish, simply by changing the genetic plans that are used to build it. Two natural enzymes Restriction enzymes and DNA ligaseare the keys to recombinant DNA technology, allowing us to edit the information in a DNA strand .

 Before the discovery of these enzymes, researchers modified the genetic code of living organisms by using biologys own tools of mating and crossing or by random mutagenesis with chemicals or ionizing radiation. Today, researchers modify the genetic code rationally at the atomic level.

 Today, recombinant DNA technology has flowered. Researchers are continually discovering new methods for harnessing the protein production machinery of the cell in new ways. Consistent methods, often in the form of commercial kits, are available for every possible process.

 We can find and extract specific genes from organisms. We can duplicate and determine the sequence of large quantities of these genes. We can mutate, recombine, and splice these genes or create entirely new genes nucleotide by nucleotide. Finally, we can replace these genes into cells, modifying their genetic information.

 DNA May Be Engineered with Commercially Available Enzymes

Restriction enzymes
Restriction enzymes are isolated from bacteria. Large number of enzymes are available commercially. Each one cuts DNA at a specific sequence of bases. Typically, restriction enzymes are composed of two identical subunits, so they attack DNA symmetrically and cut at specific palindromic sequences.

Construction of recombinant DNA

1. Fragmentation of DNA by restriction endonucleases 2. Isolation of specific human DNA 3. Insertion of human DNA into vector to form chimeric or hybrid DNA molecule 4. Joining of two different DNA fragments by DNA ligase

Fragmentation of DNA
Restriction endonucleases These are found in wide variety of prokaryotes These enzymes were originally called restriction enzymes because their presence in a given bacterium restricted the growth of foreign infective bacterial viruses

 The cells own DNA is not degraded because sequences recognised by its own restriction enzymes are methylated, there by protected from the action of restriction enzyme

Restriction endonucleases
Restriction enzymes cut DNA of any source in a sequence specific manner in contrast to action of most other endonucleases which break DNA randomly

 Restriction endonucleases recognise specific base sequences of double stranded DNA usually 4-6 base pairs in length and cleave both strands within this sequence Restriction endonucleases are named after the bacterium from which they are isolated

Nomenclature of restriction enzymes

Eco R1 Pst I : Hind III Not I : E coli Providencia stuartii : Haemophilus influenza : Norcardia otitidis-caviaru

Type II restriction enzymes

Recognition sites are palindromes AAGCTT TTCGAA GAATTC CTTAAG

Restriction endonucleases cut DNA molecules at defined positions

A restriction enzymes binds to DNA at a specific sequence and make a double-stranded cut at or near that sequence.

Odds of cutting at a segment of DNA 4 bp cutter: 44 = 256 bp 6 bp cutter: 46 = 4 kb 8 bp cutter: 48 = 64 kb How many do you predict Eco R1 to cut human genome of 2.8 x 109 bp Total No. of fragments 7x105

DNA Ligase
DNA ligase reconnects broken DNA strands. When two sticky ends anneal, DNA ligase is used to reconnect the breaks.

 DNA polymerase creates a new DNA strand by using another strand as a template, creating a double helix from a single strand. It is used to fill single-stranded gaps and to copy entire pieces of DNA.

Vectors
A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined.
Essential properties of a vector include

1) It must be capable of autonomous replication with in a host cell, 2) It must contain at least one specific nucleotide sequence recognized by a restriction endonuclease, and 3) It must carry at least one gene that confers the ability to select for the vector such as an antibiotic resistance gene. Commonly used vectors include plasmids and bacterial viruses(bacteriophages)

Plasmids
Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to host cell Plasmids have several properties that make them extremely useful as cloning vectors They replicate independently from bacterial DNA

Phages
Phages usualy have linear DNA molecules into which foreign DNA can be inserted at several restriction enzyme sites A major advantage of phage vectors is that while plasmids accept DNA pieces about 6 to 10 KB long, phages can accept DNA fragments 10-20 Kb long

Cosmids
Cosmids are plasmids that contain the DNA sequences so-called cos sites, required for packging lambda DNA into phage particles Large fragments of DNA 35 to 45 Kb can be cloned in cosmids These vectors grow in the plasmid form in bacteria but can be packaged into the phage head alongwith chimeric DNA

BIOTECHNOLOGY: VECTORS

INSERT SIZE OF VARIOUS DNA CLONING VECTORS -the choice of vector depends upon your problem
VECTOR Plasmid ** INSERT SIZE (kb) <10 kb
(see Table in Notes)

Bacteriophage **
Cosmids BACs** YACS**

9-22 kb
33-47 kb 75-350 kb 100-1000 kb

Formation of recombinant DNA

Gene cloning