Construction and screening of genomic DNA library and cDNA library

 Introduction

• DNA libraries are indeed collections of DNA fragments that have been cloned into vectors, which allow researchers to isolate and study specific genes or genomic regions. 
• Isolation of Genes: With approximately 25,000 genes in the human genome spread across 3 billion base pairs of DNA, isolating specific genes of interest is crucial for research.
• DNA Library Construction: One method for isolating genes involves constructing a DNA library. This involves cloning DNA fragments into vectors, such as plasmids or bacteriophages, to create a collection of DNA molecules representing the entire genome or a subset of it.
• Cloning: When a gene is identified and copied, it is said to have been "cloned." This process involves inserting the gene of interest into a vector, which serves as a carrier to replicate and express the gene.

Definition

• Definition of "Library": The term "library" can refer either to a population of organisms, each carrying a DNA molecule inserted into a cloning vector, or to the collection of all cloned vector molecules.
• Purpose of Libraries: DNA libraries allow researchers to identify and isolate specific DNA fragments for further study. These libraries provide ease of purification, storage, and analysis, facilitating research on individual genes and their functions.

Types of DNA Libraries

• There are different types of DNA libraries, including:
• Genomic libraries: These libraries contain DNA fragments that represent the entire genome of an organism. Genomic libraries are typically constructed by digesting the genomic DNA of an organism with restriction enzymes, which cut the DNA at specific recognition sequences, and then cloning the resulting fragments into a suitable vector, such as a plasmid or a bacteriophage.
• cDNA libraries: cDNA (complementary DNA) libraries contain DNA fragments that are complementary to the messenger RNA (mRNA) molecules present in a cell at a specific point in time. cDNA libraries are constructed by reverse transcribing mRNA into cDNA using the enzyme reverse transcriptase, followed by cloning the cDNA fragments into a vector.
• Expression libraries: These libraries contain DNA fragments that encode proteins. Expression libraries are constructed by cloning cDNA fragments into vectors that contain regulatory sequences (promoters, enhancers, etc.) to drive the expression of the cloned genes in a host organism, typically a bacterium or yeast.

Construction of Genomic Library

        There are the following main steps in gene cloning:

• Isolation of genomic DNA and vector.
• Collect DNA from an organism and prepare a small DNA carrier called a vector.
• Cleavage of Genomic DNA and vector by Restriction Endonucleases.
• Chop both the organism's DNA and the vector into small pieces using special scissors called restriction enzymes.
• Ligation of fragmented DNA with the vector.
• Mix the cut DNA pieces with the vector pieces. They'll stick together like puzzle pieces because of their matching ends.
• Transformation of r-DNA in the bacterial cell.
•  Introduce this combined DNA (called recombinant DNA) into bacteria, which act like tiny factories to make copies of the inserted DNA.

Vectors For Genomic DNA Libraries

• Genomic Libraries
• Lambda-Phase : 9 to 23kb: Convenient and easy to handle
• Cosmid: 30-45kb
• PAC, BAC, YAC : Artificial chromosomes, accommodate large fragments
• cDNA Libraries
• Lambda Phase: 9 to 23kb: Provides selection for longer cDNAs
• Conventional Plasmids : high level of expression of protein

Construction of cDNA Library

• Isolation of RNA
• Separation of mRNA from total RNA
• Synthesis of 1st strand of cDNA
• Synthesis of 2nd strand of cDNA
• Cloning of double-stranded cDNA
• Screening of cDNA Library

Let's understand the steps:

• Isolation of RNA
• Trizol Extraction
• Column Purification
• Separation of mRNA from total RNA
• Binding mRNA: 
• Oligomeric dT-coated resins grab onto mRNA molecules that have a poly-A tail, while other RNAs are left behind.
• Elution of Non-mRNA:
• RNAs without a poly-A tail are washed away, leaving only the targeted mRNA bound to the resin.
• Releasing mRNA: 
• Using an eluting buffer and a bit of heat, the mRNA is separated from the oligo-dT, allowing it to be collected for further use.
• Synthesis and Cloning
• Oligo-dT Primer Binding: 
• Oligo-dT primer binds to the poly-A tail of mRNA, creating a starting point for reverse transcriptase to make a complementary DNA strand.
• Removal of mRNA: 
• An enzyme called RNAse removes the mRNA, leaving behind a single-stranded cDNA (sscDNA) molecule.
• Conversion to Double-Stranded DNA: 
• DNA polymerase needs a free 3'-OH end to create a complementary DNA strand. The sscDNA forms a hairpin loop at its 3' end, providing the needed free end for DNA polymerase to extend. Later, an enzyme called S nuclease opens this loop.
• Cloning with Restriction Enzymes and DNA Ligase: 
• Restriction endonucleases and DNA ligase are used to insert the DNA sequences into bacterial plasmids.
• Bacterial Selection: 
• Bacteria containing the cloned DNA are selected, often using antibiotics.
• Creation of Bacterial Stocks: 
• Selected bacteria are grown and stored for future use. They can be sequenced to create a cDNA library.

Screening of Genomic Library

• Once the genomic library has been created, it is screened to identify the genes of interest. One of the most common library screening techniques is known as colony hybridization. 
• In the process of library construction, phage vectors are used when the process of identification of genes of interest involved is plaque hybridization.
• Colony hybridization:
• Preparation: 
• Create a library of DNA fragments in bacterial colonies.
• Transfer: 
• Transfer the DNA from the colonies onto a membrane.
• Denaturation and Fixation: 
• Make the DNA single-stranded and stick it to the membrane.
• Hybridization: 
• Add a labelled probe that matches the DNA sequence you're looking for.
• Washing and Detection: 
• Wash away the unbound probe and detect where the probe sticks to the DNA on the membrane.
• Identification: 
• Find the colonies where the probe stuck, indicating they contain the DNA sequence of interest.
• Plaque hybridization:
• Plaque hybridization is a technique used to identify specific DNA sequences within a population of bacteriophages, which are viruses that infect bacteria. Here's a concise explanation:
• Preparation of Bacteriophage Population: 
• Start with a population of bacteriophages, each containing DNA.
• Plaque Formation: 
• Spread the bacteriophages on a bacterial lawn, where each infected bacterial cell forms a plaque, a clear area on the lawn where bacterial cells have been killed by the phages.
• Transfer to a Membrane: 
• Transfer the DNA from the plaques onto a membrane, similar to colony hybridization.
• Denaturation and Fixation: 
• Denature the DNA and fix it to the membrane, making it single-stranded and stable.
• Hybridization with Probe: 
• Introduce a labelled DNA or RNA probe complementary to the target DNA sequence you want to detect.
• Washing and Detection: 
• Wash away the unbound probe and detect where the labeled probe binds to the DNA on the membrane.
• Identification: 
• Identify the plaques where the probe binds, indicating the presence of the target DNA sequence in those phages.
• In essence, plaque hybridization is like colony hybridization, but it's used specifically with bacteriophages instead of bacterial colonies.

Advantages of cDNA

• Stability: 
• mRNA is naturally single-stranded and prone to degradation, while cDNA, being double-stranded, is more stable. This makes cDNA a better choice for long-term storage of gene sequences.
• Representation of Expressed Genes: 
• cDNA reflects the mRNA population (genes being actively expressed) in the cell at the time of sample collection. This creates a permanent record of gene expression patterns.
• Propagation: 
• Synthesizing and cloning cDNA from a single source allows for the creation of a method to propagate the cDNA. This is facilitated by the wide range of vectors compatible with various hosts.
• Screening Complexity: 
• Both long and short cDNA molecules can be used to screen more complex genomic DNA libraries. This allows for the identification of specific genes or DNA sequences within larger collections of genetic material.
• In summary, cDNA offers advantages in terms of stability, representation of gene expression, ease of propagation, and screening capabilities compared to mRNA or genomic DNA.