part 1

Recombinant DNA Technology and DNA Cloning

Selection of Recombinant Bacteria After Transformation

The selection process is designed to facilitate the identification of recombinant bacteria from those containing plasmids without foreign DNA, while preventing the growth of non-transformed bacteria. This process is critical in identifying which bacterial cells have successfully taken up the desired recombinant plasmids containing foreign DNA.

1. Antibiotic Selection

  • In antibiotic selection, transformed cells are cultured on plates containing antibiotics to differentiate between recombinant bacteria and non-transformed bacteria.

  • This method does not select for plasmids containing foreign DNA versus re-circularized plasmids with no DNA insert.

2. Blue-White Selection Using X-gal (Artificial Lactose)

  • DNA is cloned into a plasmid's restriction site within the lacZ gene.

  • When the lacZ gene is interrupted by an inserted gene, it cannot produce functional Beta-galactosidase.

  • Non-functional lacZ results in white colonies, which represent clones of genetically identical bacterial cells each containing copies of the recombinant plasmid.

  • When X-gal is added to the media in a Petri plate, functional lacZ leads to blue colonies, indicating genetically identical bacterial cells each containing a re-circularized plasmid without the transgene.

Steps for Creating Recombinant Plasmids

  1. Isolate Plasmid (Vector) DNA and Human DNA (MCS): The gene of interest must be isolated using restriction enzymes.

    • Vector DNA containing the ampicillin-resistant gene (amp gene) is prepared using enzymes such as EcoRI and HindIII to ensure cohesive ends.

    • The plasmid should include the origin of replication and lac gene.

  2. Cut both CNAs with the Same Restriction Enzyme: The isolated human DNA needs to be inserted into the plasmids via the following steps:

    • (a) Insert human DNA into plasmids, so cohesive ends allow for joining through base pairing.

    • (b) Mix the DNAs; some plasmids will join with the gene of interest.

    • (c) Add DNA ligase to bond covalently, forming a recombinant plasmid.

  3. Introduce Plasmids to Bacteria by Transformation: The transformed bacterial cells carry plasmid DNA with the new gene insert.

Practical Applications of Recombinant DNA Technology

A. Introduction to Human Gene Cloning

  • The first recombinant human protein marketed was insulin in 1982, followed by growth hormone in 1985.

  • The technique involves cloning human insulin DNA sequence into a plasmid, and then using bacteria to synthesize the protein product of the cloned gene. This method can generate a large quantity of pure protein. The source of growth hormone prior to recombinant technology is also a point of discussion.

B. Features of a Good Vector

For effective plasmid DNA cloning, the following practical features are desired in a vector:

  • Size: The vector should be small enough to be easily separated from chromosomal DNA of the host.

  • Origin of Replication (ori): This is the site for DNA replication that allows plasmids to replicate independently of the host chromosome.

    • Copy Number: This refers to the number of plasmids in a cell; normally, recombinant plasmids have high copy numbers, allowing for greater yield of the inserted gene.

  • Multiple Cloning Site (MCS): A region containing recognition sites for several restriction enzymes into which the DNA insert is cloned.

  • Selectable Marker Genes: These markers allow for the selection of transformed colonies.

  • RNA Polymerase Promoter Sequences: These are crucial for transcription both in vitro and in vivo.

  • DNA Sequencing Primers: Primers flank the ends of the MCS to facilitate sequencing.

C. Gene Cloning Considerations

When attempting to make a lot of human insulin using a bacterial plasmid expression vector, the use of human insulin genomic DNA may not be advantageous due to the presence of introns, which complicate the cloning process.

D. Identifying and Cloning a Gene of Interest

1. Creating DNA Libraries

  • Collections of Cloned DNA Fragments: These are obtained from a particular organism and contained in plasmid vectors within host bacteria, such as E. coli. Libraries must be screened to isolate different genes of interest.

  • Two Types of Libraries:

    • Genomic DNA Libraries: These libraries are created by isolating chromosomal DNA from the tissue of interest, digesting it with restriction enzymes, and ligating the fragments into vectors. Each bacterial colony theoretically contains a single recombinant plasmid.

    • Complementary DNA Libraries (cDNA Libraries): These libraries are made by extracting mRNA from the tissue of interest, converting mRNA to double-stranded cDNA, and ligating it into vectors for bacterial transformation.

2. Disadvantages of Genomic Libraries

  • Introns, which comprise a majority of genomic DNA in eukaryotes, are cloned along with exons, resulting in a library rich in non-coding DNA.

  • Searching for the gene of interest can be time-consuming due to the large genome size in many organisms.

  • Genomic libraries do not provide information on levels of gene expression.

3. Advantages of cDNA Libraries

  • They consist of a collection of genes that are actively expressed in the cells or tissues from which the mRNA was isolated.

  • Introns are not cloned in cDNA libraries, making them less complex than genomic libraries.

  • cDNA libraries can be screened to isolate genes expressed only under certain conditions in particular tissues.

  • Disadvantage: A cDNA library can be difficult to create if a tissue source with abundant mRNA is unavailable.

E. PCR Process and Applications

  • Polymerase Chain Reaction (PCR): Developed in 1983 by Kary Mullis, PCR is a technique used to amplify a specific DNA sequence rapidly. The ingredients include target DNA, nucleotides, buffer, and DNA polymerase, along with primers.

1. PCR Cycle Stages

Each cycle consists of three stages:

  1. Denaturation: Heating to 94°C to 96°C

  2. Annealing: Cooling to allow primers to hybridize with complementary bases at about 52°C to 58°C

  3. Extension: DNA polymerase replicates the target DNA at temperatures between 70°C and 75°C.

  • Each cycle doubles the amount of DNA, and typically, 20-30 cycles are conducted.

2. Applications of PCR

  • PCR allows for the amplification of millions of copies from a small DNA sample quickly, useful in various applications like virus and bacterial infection detection, genetic diagnostics, and forensic analyses.

3. Cloning PCR Products

  • PCR products are cloned rapidly and effectively compared to traditional DNA libraries. However, knowledge of the DNA sequence flanking the gene of interest is necessary to design primers.

  • Taq polymerase, known for adding single adenine nucleotides to PCR products, aids in using T vectors for cloning.

F. DNA Sequencing Methods

  • Sanger Method (Chain Termination Method): Developed by Frederick Sanger in 1977, this technique requires a reaction tube with a single primer, dNTPs, DNA polymerase, and ddNTPs. The incorporation of ddNTPs leads to termination of the newly synthesized strands.

    • The original Sanger method involved four separate reaction tubes, isolating fragments by size using gel electrophoresis.

    • The improved high-throughput sequencing method employs capillary electrophoresis, allowing over 600 nucleotides to be sequenced per reaction, significantly enhancing speed and efficiency.

G. Applications of NGS (Next-Generation Sequencing)

  • NGS technologies such as pyrosequencing and Ion Torrent PGM utilize different biochemical processes to rapidly sequence millions of DNA fragments, greatly enhancing genomic research and applications in personalized medicine.

H. Verification Techniques

  • Techniques such as site-directed mutagenesis allow researchers to confirm specific mutations within cloned genes, vital for research in gene function and therapy development.