Essential for introducing plasmids into organisms such as E. Coli to express proteins.
In mammals, direct genome editing is required because plasmids cannot be used, e.g., modifying genes in mice or fish (e.g., Glofish).
Examples of Genetically Edited Organisms
Glofish: genetically modified fish that express colorful proteins.
Mice: genetically modified to express obesity-related genes.
CRISPR Technology
A widely-used method for genome editing.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.
Emmanuelle Charpentier and Jennifer Doudna awarded the Nobel Prize in 2020 for CRISPR-Cas9 technology.
Function of CRISPR
Acts as an immune system for Akia and bacteria against viruses by incorporating viral DNA fragments into their genomes.
Provides a defense mechanism to recognize and cut viral DNA upon re-infection.
Cas9 protein recognizes viral DNA by base pairing and functions as an endonuclease to cut precise DNA sequences.
Application in Genome Editing
Guide RNA directs Cas9 protein to specific gene sequences to be modified in laboratory organisms.
Example: Editing the FOXP2 gene in mice to observe its effects on voice phenotypes.
Genome Sequencing Techniques
Traditional Sequencing
Regular sequencing reads typically range from 500-1000 base pairs.
Example of a sequenced organism: Camphylobacter jejuni (1995), first self-replicating free-living organism sequenced, with a genome size of 1,800,000 base pairs.
Challenges in Sequencing
Traditional method requires generating new primers after sequencing each section, leading to lengthy processes (3600 weeks in example).
Shotgun Sequencing
A more efficient method that sequences random fragments of DNA.
Steps include:
Isolating and fragmenting the bacterial genome.
Analyzing fragments approximately 1.6 - 2 kb in size.
Ligating fragments to plasmids for E. coli transformations.
Sequencing using primers binding to plasmids rather than directly to genomic DNA.
Alignment Procedure
Overlapping sequences from various fragments help in assembling the entire genome sequence.
Continuous sequences (contigs) formed from overlapping reads.
Next Generation Sequencing (NGS)
Overview of Illumina Sequencing
Emerged in 2006, revolutionizing speed and cost of sequencing.
Enables millions of reads in parallel, with lower costs than previous methods.
Reduced sequence reads to approximately 15-20 bases long.
Illumina Sequencing Process
DNA fragmentation remains similar but with shorter fragments (around 50 bases).
DNA fragments are linked to a glass plate using adapters, amplifying the signal.
Sequential addition of fluorescently labeled nucleotides, with imaging after each cycle to determine the sequence based on emitted signals.
Annotation of Genomic Sequences
Moving from Sequence to Function
Identification of relevant features including open reading frames (ORFs) for proteins, tRNA genes, promoter sequences, and regulatory elements.
Finding Open Reading Frames
Analysis must be done in six frames (three on each DNA strand) due to the triplet nature of codons.
Searching for methionine (start codon) and ensuring sequences are sufficiently long (at least 30 amino acids).
Identification of codon bias for various organisms influencing their preferred codons for specific amino acids.
Basic Local Alignment Search Tool (BLAST)
Used to determine if protein sequences are found in other organisms.
Outputs alignments comparing the query sequence to known sequences with corresponding similarity metrics.
Identify conserved amino acids, indicating functional relevance across species.
Conclusion
After identifying protein-coding potential, laboratory verification includes checking for mRNA expression and protein confirmation using techniques such as mass spectrometry.
Encouragement for thorough preparation ahead of exams regarding genome sequencing and related concepts.