M03.06.02 Cloning restriction fragments of DNA: The human genome project

The Human Genome Project (HGP), initiated in 1991, sought to identify and sequence the entire 3 billion base pairs of human DNA. This monumental project, which is now complete, revealed that although humans may appear different, over 99.9% of our DNA sequences are shared between two unrelated individuals.

Key Processes in the Human Genome Project

DNA Sequencing and Cloning

The basic methodology of the HGP involved:

1. Cloning DNA restriction fragments: DNA was divided into smaller fragments using enzymes called restriction endonucleases.
2. Sequencing: The nucleotide sequence of each fragment was determined.
3. Fragment Alignment: Overlapping sequences were identified to properly align fragments and reconstruct the full genome.

Steps Involved in DNA Fragment Production

• Restriction Endonucleases: Special enzymes cut DNA at palindrome sequences, producing restriction fragments.
• Cloning in Vectors: These fragments were inserted into vectors for replication.
• Re-isolation and Sequencing: Cloned fragments were isolated and sequenced.

Importance of the Human Genome Project

The data from the HGP enables the identification of:

• Protein-coding genes
• Regulatory sequences in noncoding DNA
• Polymorphic genetic markers such as restriction sites, short tandem repeats (STRs), and single nucleotide polymorphisms (SNPs)

Restriction Endonucleases: Cutting DNA

Role in Bacteria

Restriction endonucleases are naturally found in bacteria as part of their defense system against viruses. These enzymes recognize specific palindromic sequences in DNA, which are inverted repeats of nucleotides, typically 4 to 8 base pairs in length.

Example: EcoRI Recognition Site

EcoRI, a common restriction enzyme derived from Escherichia coli, recognizes the following sequence:

• 5′ GAATTC 3′
• 3′ CTTAAG 5′

EcoRI creates “sticky ends” by cutting asymmetrically within the palindrome, which facilitates the recombination of the fragment with vector DNA.

Formation of Sticky Ends

The sticky ends produced by enzymes like EcoRI enable efficient ligation of restriction fragments with vectors during the cloning process.

Cloning DNA Fragments Using Vectors

A vector is a DNA molecule that allows autonomous replication in a host cell. Common vectors include plasmids, viral chromosomes, and yeast chromosomes. For cloning, the vector must have:

1. Palindrome sites for restriction enzymes.
2. Origin of replication for autonomous duplication.
3. Genes for antibiotic resistance, allowing for selection of successfully cloned vectors.

Example: Formation of Recombinant Plasmids

1. Cut: The plasmid and DNA fragments are cut using restriction enzymes like EcoRI.
2. Anneal and Ligate: The DNA fragment is inserted into the vector, and the ends are ligated to form a recombinant plasmid.

Genomic Libraries and Their Applications

The genomic libraries generated from projects like the HGP are used to identify various genomic elements, including:

• Protein-coding genes
• Restriction endonuclease sites
• Short tandem repeats and SNPs
• Non-expressed DNA (e.g., enhancers, promoters, introns)

Points to Remember

1. Human Genome Project sequenced 3 billion base pairs, revealing over 99.9% similarity between individuals.
2. Restriction endonucleases cut DNA at specific palindromic sequences.
3. Cloning vectors are essential for replicating and studying DNA fragments.
4. Genomic libraries enable the study of genes, genetic markers, and noncoding regions.

Bibliography

1. International Human Genome Sequencing Consortium. (2001). “Initial sequencing and analysis of the human genome.” Nature, 409, 860-921.
2. Watson, J. D., et al. (2008). “Molecular Biology of the Gene.” Pearson/Benjamin Cummings.
3. Lander, E. S., et al. (2001). “The human genome project: Decoding our DNA.” Science, 291(5507), 1304-1351.