Di-deoxy Nucleic acid sequencing: Di-deoxy and chemical sequencing methods.

Definition:

    DNA fingerprinting, also called DNA profiling, is the process of determining the precise order of nucleotides within a DNA molecule. It involves analyzing specific areas of a person's DNA that vary widely between individuals. This method is commonly used in forensic science to match crime scene evidence with suspects, in paternity testing, and in genetic studies.

    In the early 1970s, academic researchers first obtained DNA sequences using a laborious method based on two-dimensional chromatography.

Four Canonical bases are there,

• Adenine
• Guanine
• Thymine
• Cytosine

History of DNA Sequencing

• The sequencing of DNA molecules began in the 1970s with the development of the Maxam-Gilbert Method and later the Sanger-Method.
• Originally developed by Frederick Sanger in 1975, most DNA sequencing that occurs in medical and research laboratories today is performed using sequencers employing variations of the Sanger method.
• 1953 discovery of DNA double helix by James Watson and Francis Crick.
• 1965 equally Alanine tRNA was the first nucleic acid molecule to be sequenced.
• 1970 discovery of type 2 restriction enzyme by Hamilton Smith.
• 1977 Maxam Gilbert sequencing.
• 1983 polymers chain reaction developed by Kary B Mullis is a revolutionary technique that enables scientists to rapidly amplify  DNA.

Different Methods for DNA Sequencing

• Basic Methods:
• Maxam-Gilbert sequencing
• Chain termination methods.
• Next-generation methods:

• Massively parallel signature sequencing (MISS)
• Polony sequencing
• 454 pyrosequencing
• Illumina (Solexa) sequencing
• SOLID sequencing
• lon Torrent semiconductor sequencing
• DNA nanoball sequencing
• Single molecule real-time (SMRT) sequencing

Sanger Sequencing Method:

• Sanger Sequencing also known as the "Chain Termination Method" is a method for determining the nucleotide sequence of DNA.
• The Sanger sequencing method, developed by Frederick Sanger in 1977, is an efficient DNA sequencing technique. 
• It uses fewer toxic chemicals and less radioactivity than the Maxam-Gilbert method. 
• The key to Sanger sequencing is the use of dideoxynucleotide triphosphates (ddNTPs) to terminate DNA chains. 
• It requires a single-stranded DNA template, a primer, DNA polymerase, labelled nucleotides, and ddNTPs. 
• The DNA sample is split into four reactions, each with one type of ddNTP, resulting in DNA fragments of varying lengths.
• The newly made DNA fragments are heated to separate them. 
• They are then sorted by size using gel electrophoresis, with each of the four reactions running in separate lanes (A, T, G, C). 
• The DNA bands are visualized using X-ray film or UV light. 
• A dark band in a lane shows where a dideoxynucleotide (ddNTP) stopped the DNA chain. The DNA sequence is read from the bottom to the top of the gel. 
• This process can use radioactive or fluorescent labels for easier reading and faster analysis.

The building blocks of DNA and RNA.

• Adenine (A) is a base that pairs with thymine (T) in DNA and uracil (U) in RNA.
• Ribonucleotide has a ribose sugar with a hydroxyl group (OH) on the 2' carbon.
• Deoxyribonucleotide has a deoxyribose sugar with a hydrogen (H) instead of OH on the 2' carbon.
• ddNTP (dideoxyribonucleotide triphosphate) is a modified nucleotide lacking a 3' hydroxyl group, preventing further DNA strand extension, crucial for DNA sequencing.

SANGER-SEQUENCING STEPS:

• DNA SEQUENCE FOR CHAIN TERMINATION PCR
• In chain-termination PCR, the DNA sequence of interest is used as a template.
• This method works like standard PCR but includes special nucleotides called dideoxyribonucleotides (ddNTPs). 
• In standard PCR, DNA polymerase adds regular nucleotides (dNTPs) to a growing DNA strand. 
• It does this by forming a bond between the free 3’-OH group of the last nucleotide and the 5’-phosphate of the next one. In chain-termination PCR, the addition of ddNTPs stops the DNA strand from growing.
• SIZE SEPARATION BY GEL ELECTROPHORESIS
• In the second step, the chain-terminated oligonucleotides are separated by size via gel electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel matrix, and an electric current is applied; DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel. Because all DNA fragments have the same charge per unit of mass, the speed at which the oligonucleotides move will be determined only by size. The smaller a fragment is, the less friction it will experience as it moves through the gel, and the faster it will move. As a result, the oligonucleotides will be arranged from smallest to largest, reading the gel from bottom to top.
• In manual Sanger sequencing, the oligonucleotides from each of the four PCR reactions are run in four separate lanes of a gel. This allows the user to know which oligonucleotides correspond to each ddNTP.
• In automated Sanger sequencing, all oligonucleotides are run in a single capillary gel electrophoresis within the sequencing machine.
• GEL ANALYSIS & DETERMINATION OF DNA SEQUENCE
• In the final step of Sanger sequencing, the gel is read to determine the DNA sequence. DNA polymerase builds DNA in the 5’ to 3’ direction, and each ddNTP added corresponds to a specific nucleotide in the sequence. By reading the gel bands from smallest to largest, the 5’ to 3’ sequence is determined.
• Manual sequencing: The user reads the gel lanes from bottom to top, identifying each nucleotide by its ddNTP.
• Automated sequencing: A computer reads fluorescently labelled ddNTPs using a laser and produces a chromatogram showing the DNA sequence.

Applications - What are the advantages of Sanger sequencing?

• Target Smaller Genomic Regions: Effective for analyzing specific DNA regions in many samples.
• Sequence Variable Regions: Ideal for examining regions with high variability.
• Validate NGS Results: Confirms findings from next-generation sequencing studies.
• Verify Plasmid Sequences: Checks plasmid inserts and mutations accurately.
• Genotype Microsatellite Markers: Useful for genotyping microsatellite markers.
• Identify Disease-Causing Variants: Detects single genetic variants linked to diseases.

                These applications highlight the precision and reliability of Sanger sequencing for specific genetic analyses.

                                                    MAXAM-GILBERT SEQUENCE

Introduction

• Maxam-Gilbert sequencing is a DNA sequencing method developed by Allan Maxam and Walter Gilbert in 1976-1977.
• It was the first widely adopted DNA sequencing method, along with the Sanger method.
• This method involves chemically modifying DNA and then breaking it at specific nitrogenous bases.

Procedure

• Labeling: Attach a radioactive label (gamma-32P) to the 5' end of the DNA fragment.

• Chemical Treatment: Treat the DNA with different chemicals to break it at specific bases:

• Use formic acid for purines (A + G).

• Use dimethyl sulfate for guanines (G) and some adenines (A).

• Use hydrazine for pyrimidines (C + T).

• Use hydrazine with NaCl for cytosine (C). 

• Separation: Add each chemical to separate tubes.

• Fragment Generation: Generate labelled DNA fragments.

• Electrophoresis: Separate the fragments by size using electrophoresis.

• Visualization: Expose the gel to X-ray film to see dark bands, indicating the locations of the labelled DNA fragments.

Steps By Diagram:

Disadvantages of the Maxam-Gilbert Method:

• Time Consuming
• As the size of DNA increases, larger gels are required.
• Use of toxic and radioactive chemicals is hazardous.

Applications

• Understanding Functions and Diseases: Helps identify the function of specific DNA sequences and the sequences linked to diseases.
• Mutation Detection: Detects mutations through comparative DNA studies.
• DNA Fingerprinting: Used for identifying individuals based on their unique DNA patterns.
• Human Genome Project: Completing the entire human genome sequence.
• Forensics: Identifies individuals in criminal investigations using DNA from hair, nails, skin, or blood found at crime scenes.
• Agriculture: Improves crops by sequencing the genomes of microorganisms, making them more useful for farming.
• Medicine: Detects genes linked to hereditary or acquired diseases and aids in gene therapy to replace defective genes with healthy ones.