Genome and Genetic Engineering

Genome and Genes

• The genome of an organism is the total amount of genetic material (DNA/RNA) it contains, encoding information required for cell structure, function, and dynamics
• DNA molecules are long and are stored condensed in chromosomes in eukaryotes
• The size of the genome reflects the complexity of the organism
• A gene is a segment of DNA that encodes the sequence of a particular product, such as a protein

Recombinant DNA Technology

• Also known as Biotechnology or Genetic Engineering, it utilizes tissue cultures, living cells, or cell enzymes to produce specific products
• This field emerged from the ability to isolate, manipulate, and prepare small segments of DNA from larger chromosomes
• Humulin®, a recombinant human insulin, was FDA approved in 1982 as the first biotechnological agent

DNA Cloning

• Involves isolating a specific gene or segment, attaching it to a small carrier DNA molecule, and replicating this DNA many times to selectively amplify the target gene
• Requires several steps:
  • Cutting at precise locations using restriction endonucleases (restriction enzymes)
  • Joining two DNA segments using DNA ligase
  • Joining the DNA to be cloned to plasmid or viral DNA (cloning vectors)
  • Moving recombinant DNA into a host (bacteria, yeast, mammalian cell, or virus) for replication
  • Selecting and identifying host cells containing recombinant DNA and isolating the DNA

Recombinant DNA Technology Toolbox

• Restriction endonucleases: bacterial enzymes that cleave DNA at specific nucleotide sequences (restriction sites)
• DNA ligase: an enzyme that joins DNA fragments
• Cloning vectors: plasmids or viral DNA used to carry the DNA to be cloned into host cells
• Host cells: organisms used to replicate recombinant DNA

Recombinant DNA Technology Steps

• DNA is isolated from the source organism
• The target gene is identified and cut using restriction endonucleases
• The target gene is inserted into a cloning vector, typically a plasmid
• The recombinant plasmid is introduced into host cells
• Host cells are selected based on the presence of the recombinant plasmid and the gene is expressed

DNA Cleavage and Fragmentation

• Performed using bacterial enzymes that cleave DNA at specific sequences known as restriction sites
• These enzymes generate either sticky or blunt ends:
  • Sticky ends have overhangs that can base-pair with complementary sequences on other DNA fragments
  • Blunt ends have no overhangs and can be joined using a ligase

Restriction Endonucleases and Plasmid Construction

• Restriction endonucleases are used to isolate specific sequences and prepare plasmids
• The same restriction endonucleases are used to cleave both the target DNA and the plasmid
• DNA ligase joins the target DNA to the plasmid, creating a recombinant plasmid

DNA Libraries

• Collections of DNA clones used for sequencing, gene discovery, or gene function studies

• Enable the isolation of particular DNA fragments from a larger chromosome

• Two main types:

  • Genomic libraries: contain all DNA fragments from an organism, including introns and exons

  • cDNA libraries: contain only sequences that code for proteins (transcribed into mRNA)

            - mRNA of an organism is used to create cDNA using reverse transcriptase
            - mRNA is chemically destroyed 
            - A polymerase builds the complementary DNA chain
            - The cDNA is inserted into a vector and cloned, forming a cDNA library
            - Single-stranded cDNA with a radioactive species attached can be used as a probe to identify specific sequences in chromosomes
    

Polymerase Chain Reaction (PCR)

• An alternative to older cloning methods used to quickly amplify DNA sequences
• Requires knowledge of flanking sequences on either side of the target DNA
• Steps:
  • DNA is heated to denature (separate the strands)
  • Synthetic oligonucleotides (primers) complementary to the flanking regions are added
  • The DNA strands cool, and the primers anneal
  • DNA polymerase and excess deoxyribonucleotides are added, with the polymerase extending the primers and synthesizing complementary copies of the target DNA
  • The cycle of heating, cooling, and replication is repeated, leading to exponential amplification of the target DNA

Restriction Fragment Length Polymorphism (RFLP)

• Sequence differences between individuals occur around every few hundred base pairs, which creates variations in restriction enzyme sites
• These differences are referred to as RFLPs and lead to variations in the sizes of restriction fragments

Southern Blot

• A method for detecting RFLPs in DNA:
  • DNA is cleaved with a restriction enzyme
  • Fragments are separated based on size using gel electrophoresis
  • DNA fragments are transferred to a nitrocellulose nylon membrane
  • The membrane is immersed in a solution containing a radioactively labeled DNA probe, which binds to complementary sequences
  • An X-ray reveals the fragments that bound to the probe
• Southern blotting is used in forensic science, paternity tests, and prenatal genetic disease diagnosis

"Omics" Related to Recombinant DNA Technology

• Genomics: the study of genomes
• Proteomics: the study of proteins
• Transcriptomics: the study of RNA transcripts
• Metabolomics: the study of metabolites