3.

1 Construction of cDNA Library and Genomic Library

A gene library is a collection of cloned DNA fragments that together represent either
the entire genome of an organism or the specific set of genes expressed in a
particular cell type.

A. cDNA Library (Complementary DNA Library)

A cDNA library contains only the "expressed" part of the genome (exons). It is
prepared from the messenger RNA (mRNA) of a specific tissue or cell.

Procedure for Construction:

1. mRNA Isolation: Total RNA is extracted. Since eukaryotic mRNA has a poly(A) tail,
it is isolated using oligo(dT) cellulose chromatography or magnetic beads. This
removes ribosomal and transfer RNA.

2. First Strand Synthesis: The enzyme Reverse Transcriptase uses the mRNA as
a template to synthesize a complementary DNA (cDNA) strand, starting from an
oligo(dT) primer. This creates an mRNA-DNA hybrid.

3. Second Strand Synthesis (RNase H Method): RNase H is added to nick the

RNA strand in the hybrid.

o DNA Polymerase I then uses the RNA fragments as primers to replace the RNA
with a second DNA strand.

o DNA Ligase seals the nicks to create a complete double-stranded cDNA (ds-
cDNA).

4. Cloning: To insert the ds-cDNA into a vector (like pBR322 or lambda phage),
Linkers or Adaptors are often added to the ends to create compatible sticky
ends.

5. Transformation: The recombinant vectors are introduced into host bacteria (E.
coli), forming the library.

B. Genomic Library

A genomic library contains the entire DNA of an organism, including exons, introns,
promoters, and non-coding regions.

Procedure for Construction:

1. DNA Extraction: Total genomic DNA is isolated from the target organism.

2. Fragmentation: The DNA is broken into manageable pieces. This is usually done
by partial digestion with a restriction enzyme (like Sau3AI) to ensure overlapping
fragments.

3. Vector Preparation: A high-capacity vector is chosen based on the genome size:

o lambda Replacement Vectors (for inserts up to 20 kb).

o Cosmids (for inserts up to 45 kb).

 o BACs/YACs (for very large genomes).

4. Ligation: The genomic fragments are ligated into the prepared vectors.

5. Packaging/Transformation: If using lambda phage, the DNA is packaged in vitro

and used to infect E. coli. If using plasmids/BACs, the host is transformed.

Comparison: cDNA vs. Genomic Library

Feature cDNA Library Genomic Library

Source
mRNA Chromosomal DNA
Material

Enzymes Used Reverse Transcriptase Restriction Endonucleases

Introns/
Absent Present
Promoters

Size Smaller, specific to cell type Large, represents whole organism

Protein expression, studying Genome mapping, studying gene

Applications
mRNA structure

3.2 Construction of Subtractive cDNA Library

A subtractive cDNA library is a specialized tool used to identify and isolate genes
that are expressed in one specific cell type or under a certain condition (the
Target/Tester), but not in another (the Reference/Driver). This is highly effective
for studying differential gene expression, such as comparing a cancer cell to a normal
cell or a bacterial cell before and after antibiotic treatment.

The Principle of Subtraction

The goal is to "subtract" or remove all the mRNA/cDNA sequences that are common to
both cell types, leaving behind only the unique sequences.

Procedure for Construction:

1. Preparation of Nucleic Acids:

o Tester (Target): mRNA or cDNA is isolated from the cells containing the unique
genes of interest.
 o Driver (Reference): A large excess of mRNA or cDNA is isolated from the control
cells (the cells that do not express the target genes).

2. Hybridization:

o The Tester cDNA and the Driver mRNA/cDNA are mixed together, denatured (if
double-stranded), and allowed to hybridize (anneal).

o Because the Driver is added in vast excess, any sequence present in both
samples will form a Tester-Driver hybrid.

3. Removal of Common Sequences (The Subtraction Step):

o The common hybrids (Tester-Driver) and the excess Driver molecules are removed
from the mixture.

o This is often achieved using biotinylation: The Driver DNA is labeled with biotin.
After hybridization, the biotin-labeled molecules are pulled out using
streptavidin beads or phenol-chloroform extraction.

4. Recovery of Unique Sequences:

o The remaining un-hybridized molecules are the "subtracted" sequences—the ones

unique to the Tester cell.

5. Cloning and Amplification:

o These unique single-stranded cDNA fragments are converted into double-stranded

DNA using DNA Polymerase.

o They are then ligated into a vector and transformed into host cells to create the
Subtractive Library.

Applications:

 Differential Gene Expression: Identifying genes turned "on" during specific

developmental stages.

 Disease Research: Isolating genes that are over-expressed in tumors compared

to healthy tissue.

 Stress Response: Finding genes that a plant or microbe activates specifically in

response to drought, heat, or infection.

Advantages:

 It enriches the library for rare or low-abundance transcripts that would

otherwise be "masked" by highly abundant housekeeping genes (like actin or
tubulin) which are present in all cells.

3.3 Screening of Gene Libraries

Once a genomic or cDNA library has been constructed, it consists of thousands of

different bacterial colonies, each containing a different DNA fragment. Screening is
the process of identifying the specific colony that contains the gene of interest. One of
the most common methods is DNA Hybridization.
A. The Principle of DNA Hybridization

Hybridization is based on the ability of a single-stranded DNA molecule (the probe) to

bind to its complementary DNA sequence through hydrogen bonding between base
pairs (A-T and G-C).

B. The Probe

 Definition: A short, single-stranded DNA or RNA fragment that is complementary

to a portion of the target gene.

 Labeling: To visualize where the probe binds, it is labeled with either:

o Radioisotopes: Commonly 32-P, detected using X-ray film

(autoradiography).

o Fluorescent tags: Detected using specialized scanners or UV light.

 Source: Probes can be synthesized based on a known partial protein sequence

(using the genetic code) or derived from a related gene from another species
(heterologous probe).

C. Step-by-Step Procedure: Colony Hybridization

This technique allows researchers to screen colonies growing on an agar plate.

1. Master Plate Preparation: Bacteria from the library are plated on agar and
grown into distinct colonies.

2. Transfer to Membrane (Blotting): A nitrocellulose or nylon membrane is

pressed onto the surface of the agar plate. Some cells from each colony stick to
the membrane in the exact same positions as on the plate.

3. Lysis and Denaturation: The membrane is treated with chemicals (usually

NaOH) to:

o Lyse the bacterial cells.

o Denature the released DNA (convert double-stranded DNA into single-

stranded DNA).

4. Fixing: The DNA is permanently "fixed" to the membrane using heat (baking) or
UV cross-linking.

5. Hybridization: The membrane is incubated in a solution containing the labeled

single-stranded probe. The probe swims over the membrane and binds only to
the DNA of the colony that contains the matching gene.

6. Washing: The membrane is washed to remove any excess or weakly bound

probe that didn't find a perfect match.

7. Detection: If using a radioactive probe, the membrane is exposed to X-ray film

(Autoradiography).

o The "spots" or dark signals on the film correspond to the positions of the
positive colonies on the original master plate.
 8. Recovery: The researcher goes back to the original master plate, identifies the
positive colony, and picks it to grow a pure culture of the desired gene.

D. Plaque Hybridization

If the library was constructed using a Bacteriophage lambda vector, the same
process is used, but instead of bacterial colonies, the membrane picks up phage
particles and DNA from plaques (clear zones of lysed bacteria) on the lawn of E. coli.

Summary of Screening Tools

Component Function

Nitrocellulose/Nylon Provides a solid support for the DNA during the

Membrane screening process.

Denaturant (NaOH) Makes the DNA single-stranded so the probe can bind.

The "search tool" that finds the specific sequence via

Probe
complementarity.

The visualization method to locate the "hit" on the

Autoradiography
membrane.

3.4 DNA Sequencing: Maxam-Gilbert, Sanger Dideoxy, and Automated

Methods

DNA sequencing is the process of determining the precise order of nucleotides (A, T,
C, and G) within a DNA molecule. According to your document cdna and
[Link], there are two classical "first-generation" approaches and their
modern automated versions.

A. Maxam-Gilbert Method (Chemical Method)

Developed by Allan Maxam and Walter Gilbert, this method relies on the chemical
degradation of DNA.

Principle: DNA is chemically modified and then cleaved at specific bases (G, A+G, C,
C+T).

Procedure: 1. The DNA is labeled at one end (usually with 32-P at the 5' end).
2. The sample is divided into four tubes, each treated with a different chemical that
modifies and breaks the DNA at specific bases.

3. The resulting fragments are separated by size using high-resolution polyacrylamide

gel electrophoresis.

4. The sequence is read from the autoradiograph of the gel.

 Status: It is rarely used today because it uses hazardous chemicals (like

hydrazine) and is difficult to automate.

Advantages of chemical degradation method

 Sequencing by this method can be initiated anywhere in the target DNA.

 Original DNA molecule can be sequenced rather than the complementary copy.
Disadvantages of chemical degradation method

 Usually for smaller DNA fragment

 Reactions are normally slower and less consistent.
 Chemicals for cleavage reactions are hazardous.
 Labor intensive and cannot be automated.

B. Sanger Dideoxy Method (Enzymatic Method)

Developed by Frederick Sanger, this is the most widely used classical method and is
based on DNA synthesis.

 Principle: It uses "chain terminators" called dideoxynucleotides (ddNTPs).

Unlike normal dNTPs, ddNTPs lack a 3'-OH group. Once a ddNTP is incorporated into
a growing DNA strand, no further nucleotides can be added because the
phosphodiester bond cannot form.

 Procedure:

1. Reaction Mixture: Includes a single-stranded DNA template, a primer, DNA

polymerase, normal dNTPs (A, T, C, G), and a small amount of one specific
ddNTP (e.g., ddATP) per tube.

2. Synthesis: The polymerase synthesizes new DNA. Occasionally, it picks up a

ddNTP instead of a dNTP, which terminates the chain.

3. Fragment Collection: This results in a series of fragments of different lengths,

each ending at the specific base corresponding to the ddNTP used in that tube.

4. Electrophoresis: The products from the four tubes (A, T, C, G) are run in
parallel lanes on a gel.

5. Reading: The sequence is read from the bottom (shortest fragments, 5' end) to
the top (longest fragments, 3' end).

Advantages and Limitations

  Advantages: High accuracy (considered the "gold standard") and long read
lengths (typically 400-900 base pairs).

 Limitations: Not suitable for large-scale, high-throughput sequencing

compared to Next-Generation Sequencing (NGS).

 Detection Sensitivity: Cannot detect low-level mosaicity (below 15–20%) in

DNA samples

C. Automated DNA Sequencing

Modern sequencing is an evolution of the Sanger method designed for high

throughput.

 Fluorescent Labeling: Instead of using four separate tubes and radioactive

labels, each of the four ddNTPs is labeled with a different colored fluorescent
dye (e.g., ddATP is green, ddCTP is blue, etc.).

 Single Lane Reaction: All four reactions can take place in a single tube and be
loaded into a single lane or a thin capillary tube.

 Laser Detection: As the DNA fragments migrate through the gel/capillary, they
pass a laser beam. The laser excites the fluorescent tags, and a detector records
the color of each fragment.

 Chromatogram: The data is converted by a computer into a chromatogram

(a series of colored peaks), where each peak represents a nucleotide in the
sequence.

Comparison Summary (From your slides):

Feature Maxam-Gilbert Sanger Method

Type Chemical degradation Enzymatic synthesis

Double or single-stranded
Requirement Single-stranded DNA
DNA

Automation Not available Highly automated

Hazard High (toxic chemicals) Low

Key Breaks DNA at specific Terminations of chain

Mechanism bases elongation

3.5 Overview of Next Generation Sequencing (NGS) Technologies

Next Generation Sequencing (NGS), also known as high-throughput sequencing,

represents a significant leap from classical Sanger sequencing. While Sanger
sequencing reads a single DNA fragment at a time, NGS allows for the massively
parallel sequencing of millions of fragments simultaneously.

A. Key Principles of NGS

Unlike the "first-generation" Sanger method, which relies on capillary electrophoresis,

NGS platforms generally follow these core steps:

1. Library Preparation: Genomic DNA is fragmented, and specialized adapters

are ligated to the ends of the fragments. These adapters allow the DNA to bind
to a solid surface (like a flow cell or bead).

2. Clonal Amplification: To generate a signal strong enough for detection, each

individual DNA fragment is amplified in an isolated spot (e.g., through Bridge
PCR or Emulsion PCR).

3. Sequencing by Synthesis (SBS): Most NGS platforms determine the

sequence by detecting which nucleotide is incorporated as a new DNA strand is
being built.

4. Massive Parallelism: Millions of these sequencing reactions happen at the

same time on a single "chip" or "flow cell."

B. Major NGS Platforms and Technologies

Based on current industry standards and the evolution of the field mentioned in your
materials, the following are the primary technologies:

1. Illumina (Solexa) Sequencing:

 Mechanism: Uses reversible dye-terminators. All four fluorescently labeled

nucleotides are added at once. After a base is incorporated, a laser identifies the
color, the fluorescent tag is chemically removed, and the next cycle begins.

 Amplification: Uses Bridge PCR on a glass flow cell to create "clusters" of

identical DNA fragments.

 Output: High accuracy and extremely high throughput; currently the most
widely used NGS technology.

2. Ion Torrent (Ion Proton/PGM):

 Mechanism: Instead of light or fluorescence, it detects hydrogen ions ($H^+

$). When a nucleotide is incorporated into a DNA strand, a proton is released,
changing the pH of the solution.

 Detection: A sensitive "ISFET" (Ion-Sensitive Field Effect Transistor) chip acts as

the world's smallest pH meter to record the sequence.

3. Pyrosequencing (454 Sequencing):

 Mechanism: Detects the release of pyrophosphate (PPi) during nucleotide

incorporation. This PPi triggers a chemical reaction involving the enzyme
luciferase, which produces a flash of light.

 Status: While it was the first NGS technology to reach the market, it has largely
been replaced by Illumina due to cost and throughput.

C. Third Generation Sequencing (Single Molecule)

These technologies skip the amplification step and sequence a single molecule of DNA
in real-time.

 Pacific Biosciences (PacBio): Uses SMRT (Single Molecule Real-Time) cells to

observe DNA polymerase working in real-time.

 Oxford Nanopore: Measures the change in electrical current as a single

strand of DNA is pulled through a microscopic protein pore (nanopore).

D. Comparison: Sanger vs. NGS

Next Generation Sequencing

Feature Sanger Sequencing
(NGS)

Throughp Low (one read per

Extremely High (millions of reads)
ut reaction)
 Next Generation Sequencing
Feature Sanger Sequencing
(NGS)

Read Long (up to 800–1000 Often Short (50–300 bp, platform

Length bp) dependent)

Cost High per megabase Very Low per megabase

Primary Single gene, small Whole genomes, transcriptomes (RNA-

Use fragments seq)