In-Depth Notes on Recombinant DNA, Sequencing, and the Human Genome Project

Foundations: This technology utilizes DNA polymerase, restriction endonucleases, and ligases.
- DNA polymerase: Enzyme responsible for copying DNA.
- Restriction endonucleases: Enzymes that cut DNA at specific sequences.
- Ligases: Enzymes that join DNA fragments together.
DNA Cloning: Involves vectors such as plasmids or BAC (Bacterial Artificial Chromosomes) and techniques like PCR (Polymerase Chain Reaction).
Genome Mapping: Identifying the relative locations of genes by examining inheritance patterns.
- Used to create sequence maps showing order and spacing of genes in base pairs.

Genetic Markers: Variations in gene sequences that can be identified and used for genetic mapping.
- Polymorphic Alleles: Alternative forms of genes that exist at certain loci.
Restriction Fragment Length Polymorphisms (RFLPs): Differences in DNA sequences that result in varying lengths of DNA fragments after digestion with restriction enzymes.
Simple Sequence Length Polymorphisms (SSLPs): Variations in length of short repeating sequences in DNA (e.g., 2-25 bp).
Single Nucleotide Polymorphisms (SNPs): A single base pair variation in a specific gene region among individuals.

Sanger Method: Also known as the chain-termination method, developed in 1974, using dideoxynucleotide triphosphates (ddNTPs) to terminate DNA synthesis. Awarded Nobel Prize in Chemistry in 1980.
Shotgun Sequencing: A method that fragments DNA into smaller pieces, which are then sequenced and assembled again.
- Uses restriction endonucleases to create smaller fragments for sequencing and assembly.

Launched in 1990 and aimed to determine the sequence of the haploid human genome.
- Completion: First draft published in 2001, with final completion in 2003 and last chromosome finished in 2006.
- Cost: Approximately $3 billion.
- The reference genome was a composite from various donors.

In 1998, Celera Genomics led by Craig Venter aimed to finish sequencing the genome sooner (3 years) for around $300 million, with intentions to patent identified genes.
Challenges arose as public announcements placed stress on Celera’s stock and competitive pressure.
Both projects announced the draft sequence together in March 2000.

The human genome comprises approximately 3 billion base pairs with around 20,000-25,000 genes. Only about 1.1-1.4% of the genome encodes proteins.
Key problematic regions remain unsequenced and consist mainly of repetitive DNA (centromeres and telomeres).

Genomic data is stored in databases like NCBI and Ensembl, with tools for data annotation and visualization.
Minimum Tiling Path (MTP): Refers to the least number of BACs needed to cover the sequence completely.

Adopted by HGP: A mapping strategy that sequences chromosomes in parts, creating a physical map before sequencing.
Involves creating a BAC library, mapping the BACs, and sequenced parsing them into smaller DNA pieces for assembly.

Coverage: The average rate at which individual nucleotides are read (the example shows a coverage of 7.5X based on sequencing data).
WGS: Utilizes a shotgun approach skipping the mapping phase but requires high coverage (about 20X) for accurate assembly.

Major issues include sequence errors, contamination, and repeats.
- Tools developed during HGP such as PHRED and PHRAP assist in sequence correction and assembly.
PHRED: A base caller that converts raw sequencing data into sequences with quality scores, indicating reliability.
PHRAP: A sequence assembler that aligns sequences based on quality scores to improve accuracy during sequencing assembly.