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Introduction

• Genetic information use is commonplace for scientists
• Using genetic information as a tool requires knowledge of navigation inside the whole genome
• The human genome is about 3.3 x 10^9 Kbp
• The Human Genome Project is a project to understand the human genome

Generating Genomic Information

• Genomic information can be obtained through a genomic library
• Genomic libraries are a collection of cloned sequences that represent the entire genome including both exons and introns
• Genomic libraries are used for the analysis of gene promoters affecting gene functions and regulation
• Genomic libraries are used for the creation of maps of the genome locating exact genes and their relative distances from each other
• Human Genome Sequencing Project relies on genomic libraries extensively
• Complementary DNA (cDNA) libraries contain information that is expressed from the genome
• cDNA libraries contain only exonic sequences

Tools for Genomic and cDNA Libraries

• Enzymes include:
• Restriction enzymes
• I DNA polymerase
• Reverse transcriptase
• Terminal transferase
• DNA ligase
• DNA methylase
• Vectors include:
• Plasmids
• Bactriophage Lambda (λ phage)
• Yeast Artificial Chromosome (YAC)
• Bacterial Artificial Chromosome (BAC)

Restriction Enzymes

• Enzymes that can recognize specific sequences in DNA and break both strands inside or near to the recognition site
• Require Mg2+
• EcoRI, Pstl and Smal are examples of restriction enzymes
• EcoRI generates 5' overhangs
• Pstl generates 3' overhangs
• Smal generates blunt ends

Restriction Endonucleases

• Restriction endonucleases are crucial components of the bacterial defense system
• The restriction/modification system is used by many bacterial species
• These enzymes prevent foreign DNA (like viruses) from infecting the host by breaking it down
• Host DNA has methylation to prevent degradation by the bacterial defense system
• Methyl groups are added to cytosine or adenine nucleotides

Reverse Transcriptase

• mRNA can have reverse transcription performed, via a DNA primer
• The result is 1st strand cDNA

DNA-dependent DNA polymerase

• DNA primase, DNA ligase uses Mg
• Some examples also use Klenow fragment

Terminal Transferase

• Terminal transferase adds deoxynucleotides to the DNA molecule's 3' hydroxyl terminus
• It is a template-independent DNA polymerase
• This can be used to add homopolymeric tails or single nucleotides to DNA's 3' strands

DNA Ligase

• DNA ligase can form new 3-5 phosphodiester bonds

Vectors

• Vectors are vehicles used to transfer genetic material into target cells
• Vector characteristics:
• Origin of replication
• Selection marker
• Multiple cloning site
• Capacity

Plasmid

• Plasmids are extra-chromosomal DNA molecules separate from chromosomal DNA
• Plasmids can replicate independently of chromosomal DNA
• Plasmids usually occur naturally in bacteria, and some are located in eukaryotic organisms like Saccharomyces cerevisiae
• Plasmids are circular and double-stranded
• Plasmids in molecular biology:
• Relatively small molecular weight
• Have a drug resistance gene
• Multiple cloning site
• Origin site for replication
• Plasmid applications:
• Cloning
• Sequencing
• In vitro transcription
• Gene expression to produce protein in bacteria

λ-phage as a Vector

• λbacteriophage viral particles contain a head and tail that has tail fibers
• The head contains 48,490 base pairs of double-stranded, linear DNA with 12-base-pair, single-stranded segments forming the cos site
• The phage genome loops into a circle in the host cell cytoplasm and so is 48,502 base pairs in length
• Prophage exists as a linear section of DNA inside the host chromosome
• λ-phage is either lytic or lysogenic
• The cloning capacity is 23 Kbp insert

Lytic vs. lysogenic cycle of the λ-phage

• Lytic - relating to lysis, typically kills the host cell after replicating
• Lysogenic - viral DNA incorporated into host DNA
• Both start with viral attachment to a host cell

Molecular Events Leading to the Lysogenic Cycle

• A virus particle's linear DNA circularizes attaching to a bacterial cell to attach cos sites
• Recombination integrates this molecule into the bacterial chromosome

Yeast Artificial Chromosome (YAC)

• Vector with yeast sequence for chromosomal DNA replication including:
• TEL: segment for telomeric DNA sequence
• CEN 4: centromere sequence
• ARS: autonomously replicationg sequence (similar to origin of replication)
• Selection of recombinants
  • TRP1, URA 3: yesat selectable markerks
    • SUP 4: The yeast SUP4 gene contains a cloning site used as a colour marker for selection of YACs containing exogenous insert DNA. SUP 4 causes the accumulation of red pigment
• Has a cloning capacity: of 0.5-2 Mbp
• Recombinant YAC are propagated in Saccharomyces cerevisiae

Bacterial Artificial Chromosome (YAC)

• BAC vectors are similar to standard E. coli plasmid vectors.
• BAC vectors contain the origin of replication and genes encoding the ORI-binding proteins required for plasmid replication
• BAC vectors have low copy numbers typically 1-2 copies per cell Transformation occurs by electroporation of recombinant DNA into E. coli
• BAC vectors can accommodate DNA fragments up to 300 Kbp