Molecular Cloning or Genetic Engineering or Recombinant DNA

Molecular Cloning or Genetic Engineering or Recombinant DNA Technology: To clone means to make identical copies. DNA cloning involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small carrier DNA. The resultant hybrid DNA is called recombinant DNA, which is transferred to a proper host (bacteria, virus or yeast) and replicated to make multiple copy of the selected gene. When cloned under an appropriate expression vector, a gene can be expressed (I.e. transcribed and translated), at desired level to produce recombinant proteins. This technology has made it possible to isolate, clone and produce DNA for all the genes in appropriate quantity so that they can be sequenced and characterized. Similarly, some of the genes which are expressed at very low level, can be cloned and desired amount of recombinant proteins can be produced.

Five steps of cloning: 1. Cutting the DNA to be cloned from the chromosomal using sequence specific Restriction Endonuclease. 2. Selecting a cloning vector (a small molecule capable of selfreplicating inside host cells), and cutting the cloning vector with the same restriction endonuclease producing the cohessive ends. 3. Incubating the vector and subject DNA togather to aneal and then joining them using DNA ligase. The resultant DNA is called recombinant DNA. 4. Transferring the reconbinant DNA to an appropriate host such as bacteria, virus or yeast which will provide necessory biomachinary for DNA replication. 5. Identifying the host cells that contain the recombinant DNA.

Cloning Vectors: Circular plasmid DNAs are the most common cloning vectors. These are 1 to 200 kb long DNA duplexes containing required genetic machinery such as replication origin to permit their autonomous propagation in host cell. The plasmid vectors contain some specific genes responsible for antibiotic resistance, which can be used to select the bacterial colonies containing recombinant plasmids. In order to clone the foreign DNA at specific site, a synthetic oligonucleotide containing restriction sites for several REs is ligated in the plasmid. This region is reffered as polylinker region. Bacteriophage-based cloning vectors Yeast artificial chromosomes vectors

Joining of two DNA fragments: Dale Kaiser and Paul Berg: Used terminal deoxynucleotidyl transferase (TdT or terminal transferase) to generate sticky or cohesive ends in the DNA. TdT is a mammalian enzyme which adds nucleotide to the 3’-OH group of DNA without any requirement of primer. The two DNA fragments to be joined, are subjected to TdT reaction in the presence of dTTP and dATP separately to add poly-T to one DNA and poly-A to other DNA. The two DNA fragments with cohesive ends are annealed, the gaps are filled with DNA polymerase and then they are joined covalently by ligase. TdT requires at least three nucleotides free at 3’ end (I.e unpaired), it can be created by bacteriophage lambda exonuclease.

A constructed E. Coli plasmid pBR322 designed specially for cloning in E. Coli. A foreign gene cloned in PstI restriction site can be selected as depicted in next slide.

Selection of the bacterial colony containing recombinant DNA by antibiotic resistance and sensitivity.

Bacteriophage cloning vector: This virus is very efficient in delivering its 48kb long DNA into a host bacterium. One third of its DNA is nonessential and can be replaced by foreign DNA. The recombinant DNA can be packaged into phage particle by adding this DNA to bacterial extract containing proteins for packaging.

Construction of complementary DNA (cDNA) library: cDNAs are the DNA with complementary sequence to mRNA. The cDNA represents genes expressed at mRNA level. 1. 2. 3. 4. mRNA is isolated using oligi-dT column and annealed with oligodT primer. cDNA is generated by reverse transcriptase and dNTPs The mRNA is degraded by alkaline hydrolysis and a double standed DNA is prepared using DNA polymerase-I and dNTPs. The cDNAs created in this way are cloned in appropriate plasmid or phage vector and tranfected to host cells.

Amplification of a DNA segment by Polymerize Chain Reaction (PCR): 1. 2. 3. 4. DNA strands are separated by heating. Cool the DNA and add synthetic oligonucleotide primers that flank the region to be amplified. Add thermostable DNA polymerase (Taq1 polymerase) to catalyse 5’-3’ synthesis of DNA. Repeat steps 1, 2 and 3 30 to 40 times to generate thousands to millions of copies of the original DNA.

Colony –hybridization to screen the bacterial plasmid library: 1. The cDNA library (bacteria containing different cDNAs) is plated on agar plates in appropriate media. 2. A nitrocellulose paper is pressed upon the the bacterial colonies. Some bacteria are transferred to NC paper. 3. The NC is treated with alkali to lyse the cells and expose the cDNAs. 4. The DNA binds to NC paper, and a radioactive DNA probe corresponding to the desired gene is used to hybridize with the NC paper. 5. DNA from the Colonies with the desired gene will be seen on X-ray film after the exposure of the hybridized NC paper.

DNA Microarray analysis for gene expression: 1. DNA chips conatining spots with the DNA of known genes are available commercially, or one can make one with desired DNAs. 2. mRNA is isolated from control and diseased tissue and cDNA is made using different fluorescent nucleotide for the two mRNAs. 3. The fluorescent cDNAs is then used for hybridization with the DNA microarray chip. 4. The fluorescent spots indicate the expression of corresponding gene.

Site-directed Mutagenesis: Michael Smith (Canada) was awarded with noble prize for his work on site directed mutagenesis. 1. One can synthesize a mutated DNA and insert into the gene using restriction enzyme and ligase. 2. The most poular proceedure involves working with single stranded DNA. An oligonucleotide is made with desired single nucleotide change and then used as a primer with DNA polymerase to make a mutant copy of genes.

Creation of recombinant plant using a plant parasite agrobacterium and two plasmid stretegy.

A tobacco plant in which the gene for fire fly luciferase is expressed: the plant glows when watered with luciferin (a substrate for this enzyme).

Tomato plants engineered to be resistant to some insect larvae