UNIT 8. DNA SEQUENCING

cDNA

• DNA synthesized from a single stranded RNA

• catalyzed by reverse transcriptase enzyme from retroviruses (e.g. HIV)

• more common and efficient way because it does not contain introns unlike gDNA

1.Ethanol precipitation

2.Phenol-chloroform extraction

3.Minicolumn purification

3 DNA Isolation Techniques

Ethanol precipitation

3 DNA Isolation Techniques

• usually by ice-cold ethanol or isopropanol.

• Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate.

Phenol-chloroform extraction

3 DNA Isolation Techniques

• phenol denatures proteins in the sample.

• After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution.

• toxic reagents

Minicolumn purification

3 DNA Isolation Techniques

• that relies on the fact that the nucleic acids may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt concentration of the buffer.

dNTPs can undergo chemical modification such as methylation, hydroxylation, and oxidation, forming a carboxylic acid

What is the challenge with dNTPs in DNA sequencing

• Maxam-Gilbert Sequencing

• Sanger Sequencing

Early DNA Sequencing Methods

Maxam-Gilbert Sequencing

Early DNA Sequencing Methods

• Radioactive label at the 5'-end

• And then chemically treat the DNA to break off 1 or 2 bases at a time.

• Visualization was done by gel electrophoresis (PAGE)

• Difficult, time-consuming, toxic chemicals, and was NOT high accuracy.

Sanger Sequencing Method/ Chain Termination Sequencing

Early DNA Sequencing Methods

• DNA sequencing method based on selective chain termination

• etermines the nucleotide order by incorporating fluorescently labeled chain-terminating ddNTPs during DNA synthesis, separating the resulting fragments by capillary electrophoresis, and detecting their terminal bases through laser fluorescence.

ddNTPs (dideoxynucleotide triphosphates)

• Modified bases with no 3' OH group, labeled with different fluorochromes for each base (A, T, G, C)

• ause chain termination because no further nucleotide can be added once they’re incorporated.

1. Reaction mixture

2. Primer elongation & chain termination

3. Capillary gel electrophoresis separation of DNA fragments

4. Laser detection of fluorophores & computational sequence analysis

5. Chromatograph

Steps in Sanger Sequencing

Microfluidics

• the science of manipulating very small volumes of fluids (microliters to picoliters) in tiny channels, often thinner than a hair.

• It allows precise control of fluid movement, mixing, and reactions on a miniature scale.

• requires less sample, less reagent, and gives faster results.

Lab-on-a-chip (LOC) Technologies

• devices use microfluidics to integrate multiple laboratory processes—such as sample preparation, reaction, separation, and detection—onto a single small chip.

• Faster

• Portable

• Cost-effective

• Suitable for point-of-care testing

• Illumina Sequencing

• Roche 454 Sequencing

• Ion Torrent Technology

Next Generation Sequencing

Illumina sequencing

Next Generation Sequencing

1. Genome fragmentation (100–150 bp reads)
   The DNA is broken into short fragments (typically 100–150 base pairs). Adapters are attached to both ends to allow binding to the flow cell.

2. Binding to the flow cell (not tiny wells)
   The fragments attach to a glass slide called a flow cell, which contains oligonucleotides complementary to the adapters.
   These fragments undergo bridge amplification, forming clusters of identical copies. Each cluster originates from a single DNA fragment.

3. Addition of fluorescent nucleotides and DNA polymerase
   The flow cell is flooded with DNA polymerase and four fluorescently labeled reversible terminator nucleotides (A, T, G, C).

4. Terminator incorporation (one base at a time)
   Only one nucleotide can be incorporated per cycle because each nucleotide has a reversible terminator that blocks further extension.

5. Imaging
   A camera captures the fluorescent signal emitted from each cluster. The color corresponds to the base added.

6. Terminator removal and repeat cycle
   The terminator and fluorescent label are chemically removed, allowing the next cycle to proceed.

7. Same-length reads
   Since one base is added per cycle across all clusters simultaneously, all fragments are extended one base at a time, making reads uniform in length.

Illumina sequencing

Next Generation Sequencing

• uses bridge amplification and reversible fluorescent terminators to determine DNA sequences one base at a time through repeated imaging cycles.

Roche 454 Sequencing

Next Generation Sequencing

1. DNA fragments are attached to beads and amplified using emulsion PCR using adaptor-specific primers

2. Each bead is placed in a tiny well of a slide.

3. Nucleotides are added one at a time.

4. When a nucleotide is incorporated, pyrophosphate (PPi) is released.

5. This triggers a chemical reaction producing light, which is detected.

Roche 454 Sequencing

Next Generation Sequencing

• Longer read lengths than early Illumina (~1 Kb)

• sequencing multiple reads at once via reading optical signals (light) as bases are added

• DNA varies in length
  
  

Disadvantages:

• Difficulty reading long homopolymer regions (e.g., AAAA)

• Expensive

• Discontinued commercially

Ion Torrent sequencing

Next Generation Sequencing

1. DNA is fragmented and amplified on beads (emulsion PCR).

2. Beads are placed on a semiconductor chip.

3. When a nucleotide is incorporated, a hydrogen ion (H⁺) is released.

4. The release changes the pH, which is detected electrically (no fluorescence).

• Faster sequencing time

• No need for fluorescent labels or cameras

• Lower instrument cost

30, 000 bases

Ion torrent technology uses a small USB that can read _________, however, is less accurate than the others.

Artificial Gene synthesis (DNA Printing)

Artificial gene synthesis is the process of chemically creating a DNA sequence from scratch, without using a natural DNA template. Instead of copying existing DNA (like PCR), scientists design the sequence on a computer and then chemically build it base by base.

1. Detritylation

2. Coupling

3. Capping

4. Oxidation

Steps in Artificial gene synthesis

Bioinformatics

•Collect statistics from biological data

•Build a computational model

•Solve a computational modeling problem

•Test and evaluate a computational algorithm

Bioinformatics

•Types of biological information and databases

•Sequence analysis and molecular modeling

•Genomic analysis

•Systems biology

Genomics

• is the study of an organism’s entire genome — all of its DNA, including genes and non-coding regions.

• It focuses on:

  • DNA sequencing

  • Gene structure and function

  • Gene regulation

  • Genetic variation

• elps us understand diseases, evolution, personalized medicine, and biotechnology.

Comparative Genomics

• compares the genomes of different organisms to identify:

  • Similar genes

  • Evolutionary relationships

  • Conserved sequences

  • Genetic differences

• scientists can determine how species evolved and identify genes responsible for specific traits or diseases.

Proteomics

• is the large-scale study of all proteins (the proteome) produced by a cell, tissue, or organism.

• It focuses on:

  • structure

  • function

  • interactions

  • Post-translational modifications

Nutrigenomics

is the study of how nutrition affects gene expression and how genes influence the way the body responds to nutrients.

Cloning Vectors

• are DNA molecules used to carry foreign DNA fragments into a host cell (usually bacteria) so the DNA can be replicated and sometimes expressed.

• commonly as plasmids, bacteriophages, and artificial chromosomes

1. Origin of Replication (Ori)

2. Multiple cloning site (MCS) / Polylinker

3. Selectable Markers

Components of cloning vector

Origin of Replication (ori)

Components of cloning vector

• is a specific DNA sequence where replication begins.

• Allows the vector to replicate independently inside the host cell.

• Ensures that the inserted gene is copied every time the cell divides.

Multiple Cloning Site (MCS) / Polylinker

Components of cloning vector

• is a short DNA region containing several unique restriction enzyme recognition sites.

• Allows insertion of foreign DNA using restriction enzymes.

• Provides flexibility because different enzymes can be used.

Selectable Markers

Components of cloning vector

• are genes that allow identification of cells that successfully took up the vector.

• Antibiotic resistance genes

  • Ampicillin resistance (ampR)

  • Kanamycin resistance (kanR)

• After transformation, cells are grown on antibiotic-containing media.

• Only cells containing the plasmid survive.

Antibiotic resistance genes

Most Common Type of Selectable markers

Palindrome

a sequence that reads the same in the 5′ → 3′ direction on both strands (when complementary base pairing is considered).

5′-GAATTC-3′
3′-CTTAAG-5′

Restriction enzymes

______________ bind to palindromic DNA as two identical units and cut both strands at specific positions, breaking the DNA backbone.

Bacillus amyloliquefaciens strain H

BamHI was isolated from the bacterium:

Buts after the first Guanine base

5′-G↓GATCC-3′
3′-CCTAG↑G-5′

BamHI cuts where?

sticky ends with 5’ overhang (GATC)

BamHI produces what type of cuts

Reporter Genes

• are genes attached to a regulatory sequence or gene of interest to indicate whether that gene is being expressed.

• They produce an easily measurable signal, such as color, fluorescence, or luminescence.

Green fluorescent protein (GFP)

Common Reporter Genes

• Produces green fluorescence under UV/blue light.

lacZ

Common Reporter Genes

• Encodes β-galactosidase, which turns X-gal blue (used in blue-white screening).

Luciferase

Common Reporter Genes

• Produces light in the presence of its substrate (luciferin).

Plasmids

Types of Cloning Vectors

• small extrachromosomal DNA

• can replicate independently

• Small, circular double-stranded DNA molecules naturally found in bacteria, or archea

• carry genes for survival and advantage of the organism (e.g. antibiotic resistance)

Bacteriophage

Types of Cloning Vectors

• viruses that infect bacteria & archaea

• more than 131 bacteriophage in the planet

• Example is lambda phage

• DNA packaged into viral particles

• Efficient infection of host cells

Cosmids

Types of Cloning Vectors

• Hybrid vectors combining plasmid features with bacteriophage λ cos sites.

• Can be packaged into phage particles

• Replicate like plasmids in bacteria

• Used for large DNA fragment cloning

BACs (Bacterial Artificial Chromosomes)

Types of Cloning Vectors

• Engineered plasmids based on the F plasmid of bacteria.

• Very stable

• Low copy number

• Suitable for large DNA fragments

YACs (Yeast Artificial Chromosomes)

Types of Cloning Vectors

• Vectors that replicate in yeast cells such as Saccharomyces cerevisiae.

• Contain yeast centromere, telomeres, and ori

• Behave like real chromosomes

• Cloning very large DNA fragments

HACs (Human Artificial Chromosomes)

Types of Cloning Vectors

• Synthetic chromosomes designed to function in human cells.

• Contain centromere, telomeres, replication origins

• Can carry very large genes

Dolly the sheep in 1996.

The first successfully cloned mammal was ___________________

Reproductive cloning

• is the process of creating a new organism that is genetically identical to another (multicellular) organism.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

is the process of creating a new organism that is genetically identical to another organism.

bacterial antiviral defense mechanisms

Origin of CRISPR

1. Guide RNA (gRNA)

2. Cas9 enzyme

3. PAM sequence (Photospacer Adjacent Motif)

Components of CRISPR-Cas9

gRNA

Components of CRISPR-Cas9

• Designed to match a specific DNA target.

• Guides Cas9 to the exact DNA sequence.

Cas9 enzyme

Components of CRISPR-Cas9

• Acts as molecular scissors.

• Cuts double-stranded DNA at the target site.

PAM sequence (Protospacer Adjacent Motif)

Components of CRISPR-Cas9

• Short DNA sequence required for Cas9 binding.

• Ensures proper targeting.

1. gRNA binds to the target dsDNA

2. Cas9 creates a double strand break in the dsDNA

3. Cell repairs break either through

   • NHEJ (Non-Homologous End Joining) - dresults to deletions/insertions

   • HDR (Homologous-Directed repair) - allow insertion or correction of genetic material

Steps of CRISPR

Nobel Prize Award in Chemistry (2020) to Jennifer Doudna and Emmanuelle Charpentier.

Nobel Prize won by CRISPR

• Gene therapy (e.g., sickle cell disease & β-thalassemia)

• Cancer immunotherapy

• Functional genomics

• Agriculture improvement

• Disease modeling

Applications of CRISPR

Microarray

• laboratory tool used to measure the expression of thousands of genes at the same time or to detect specific DNA sequences in a sample.

• based on the principle of complementary base pairing (A–T and C–G).

• a small solid surface (usually a glass slide or silicon chip) that contains thousands of microscopic DNA spots arranged in a grid pattern.

probes

Each spot in the microarray chip contains many copies (10-12 moles) of a known DNA sequence called _________

1. Isolation of mRNA

2. mRNA —→ cDNA using RT enzyme

3. cDNA is labeled with fluorophores

   • Red = diseased

   • Green = normal

4. Hybridization in the microarray chip

5. Washing of unbound sequences

6. Laser scanning & Imaging

   • Bright signal = high gene expression

   • Weak signal = low gene expression

   • Yellow (red + green) = equal expression

Steps in Microarray

• Cancer research (tumor profiling)
• Drug development
• Disease diagnosis
• Pharmacogenomics
• Toxicology studies
• Genotyping

Applications of Microarray

In situ hybridization (ISH)

• is a laboratory technique used to detect specific DNA or RNA sequences directly within cells or tissue sections, while preserving their original location

• allows us to see where a gene is located or expressed inside tissues.

• Shows location of the gene

• Maintains tissue structure

• Provides spatial information

1. Tissue preparation (fixation w formaldehyde)

2. Permeabilization

3. Probe Hybridization

4. Washing

5. Detection

Steps of In Situ Hybridization

DNA In Situ Hybridization

Types of In Situ Hybridization

• Gene mapping

• Detecting chromosomal abnormalities

• Detects specific DNA sequences in chromosomes.

RNA In Situ Hybridization

Types of In Situ Hybridization

• Which cells express a gene

• Level and pattern of gene expression

• Detects mRNA expression.

• temperature

• salt

• detergent concentration

Solution parameters in ISH that can be manipulated

1. radioactive —→ autoradiography

2. fluorophores —→ fluorescence microscopy

3. antigen —→ immunohistochemistry

ISH Probes are labeled with? and detected using?