Cot1/2 Analysis: DNA Reassociation & Genome Complexity

Genome Analysis

• Genome analysis involves determining the complete genetic information of an organism.
• The human genome comprises the complete sequence of all 46 human chromosomes, approximately 3 x 10⁹ base pairs.
• Sequencing the human genome was a significant technological achievement spanning from October 1990 to April 2003.
• Genome analysis provides valuable insights into the proteins an organism synthesizes.
• Comparative analysis of genomes, along with advanced computational tools, improves interpretation of genomic data.
• Numerous complete genomes are now available, facilitating comparative studies.

The C-Value Paradox

• The C-value paradox describes the seemingly inconsistent relationship between the size of an organism's genome and its complexity.
• The amount of DNA differs widely among eukaryotes, not directly correlating with organismal complexity.
• Example: Homo sapiens have a genome of ~1600 Mbp while Paris japonica has a genome of 150,000 Mbp, even though the complexity of Paris japonica is considered low comparatively.

Representative Genomes

• A table details the total DNA content (base pairs), chromosome count, and gene number for several organisms, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, etc.
• The table also reports the genome size of a plant species, Paris japonica.
• The data demonstrate significant variation in genome size across different organisms.

Cot1/2 Analysis

• This technique measures the complexity of a DNA sample by determining the amount and type of repeated DNA sequences present.
• The process involves randomly fragmenting double-stranded DNA.
• The DNA is separated into single strands using heat, allowing reassociation by slow cooling.
  • Varying DNA concentrations (Co) and time frames (t1/2) are used during slow cooling.
• The reassociation rate to double strands is determined by measuring hyperchromicity (UV absorbance) which significantly drops as the DNA pairs.
• The rate of reassociation is inversely related to the length of the DNA sample.

DNA Can Be Denatured By Heating

• DNA denaturation occurs with heat, causing hydrogen bonds between nucleotides to break, thus separating the two strands.
• Higher heat leads to higher percentage of single-stranded DNA.
• Interaction between electron clouds lowers UV absorbance stabilizing the helix from this separated state.
• Melting curve measures the absorbance changes of UV with increasing temperature.
• The melting temperature (Tm) is the temperature at which half of the DNA is in the single-stranded form.

DNA Fragments Can Be Denatured/Renatured: Hybridization

• Complementary DNA strands bind each other during slow cooling.
• Hybridization takes place even in DNA mixtures with various sequences.

Rate of Reassociation

• The rate at which DNA fragments renature is inversely proportional to the length of the fragments.
• shorter fragments reassociate quickly, whereas longer segments require more time.
• Shorter segments (e.g., homopolymer) recombine significantly faster.

What Kinds of Sequences Are in the Human Genome?

• Prokaryotic and viral genomes have genes organized densely along the DNA.
• Eukaryotic genomes contain far less "gene" sequences and are diverse, encompassing various duplicated sequences.
  • Simple sequences (2-12 nucleotides repeated)
  • Short interspersed sequences (SINES, 100-700 bp, ~10⁵ copies), such as Alu elements in humans.
  • Long interspersed sequences (LINES, 6000 bp, ~10⁴ copies).
  • Highly expressed genes present in multiple copies (e.g., rRNA genes present in numerous copies to support ribosomal production for protein synthesis).

The E. coli Genome

• E. coli has one circular chromosome with a single origin of DNA replication.
• It was sequenced completely in 1997 and has high quality data.
• The genome comprises 4.6 Mb/4,288 genes.
• It has closely located genes with little space for intergenic regions.
• Genes are often organized in operons, and it has limited repeated genes and no introns; some genes arose through horizontal gene transfer.

The Overall Structure of the E. coli Genome

• The origin and terminus of replication are displayed as green lines on the structure of the E.coli genome.
• Replichore regions are indicated by blue arrows from the origin/terminus of replication.
• A scale indicates coordinates in base pairs and minutes.
• Genes are depicted on the outer rings (orange boxes/genes on leading strand, yellow boxes/genes on lagging strand.
• Transcription direction is indicated by red arrows, particularly for rRNA and tRNA genes, shown as green arrows.
• CAI (Codon Adaptation Index) is a histogram of how often a given codon is used in a particular genome, and compared with a 'normal' E. coli, a histogram to show high/low expression genes.

So How Do You Find the Genes?

• Genes are identified by sequence comparisons with other organisms. Some regions of the DNA are identified as genes based on presence of open reading frames of at least 100 amino-acids.
• An ORF (open reading frame) begins with an ATG codon and ends with a stop codon (TAA, TGA, or TAG). Stop codons are expected about once every 21 triplets, out of 64 triplets.
• These methods do not encompass all gene types.

How Much of Our Genome Is Open Reading Frames?

• The human genome size comprises 3 x 10⁹ base pairs.
• If the entire sequence encoded proteins, it could yield about 2 million proteins.
• However, the human genome contains approximately 20,000 genes, covering only ~1.5% of the genome or fewer genes depending on the method used.

Repetitive and Unique Spacer DNA Sequences

• Approximately 50% of the DNA outside of genes consists of repetitive DNA sequences (multiple copies of the same sequence).
• The other 25% of spacer DNA is unique and has unknown functions.

Repetitive Spacer DNA: Tandem Repeats

• Repetitive sequences occur in tandem in a particular region.
• Tandem repeats can be direct or inverted.

Simple Repeats in the Human Genome

• Simple repeats are short sequences (1-30 nucleotides) repeated many times.
• These repeats include mononucleotide, dinucleotide, and trinucleotide repeats.
• Changes in the number of trinucleotide repeats can cause various diseases.
• Repeats are often found in non-functional regions of the genome, but can cause disease if they occur in a coding region.

Simple Repeats in Human Diseases

• Trinucleotide repeats, like CAG or CCG, result in disorders such as Huntington's disease.
• Length of trinucleotide repeats varies greatly between individuals; these are polymorphic; they are used to compare people, due to these variations in length.

Other kinds of repeats: Transposon-related elements

• Historically called "junk DNA," transposable elements encompass DNA sequences that can move around the genome through mechanisms that may be cut-and-paste or copy-and-paste.
• These elements include DNA-only and retrotransposons.

LINES: Long Interspersed Sequences

• LINES are the most abundant class of repeated sequences in humans, comprising over 20% of the genome.
• LINE sequences (e.g., L1), often vary from 1 to 9 kb in length, particularly L1 (~6.5kb).
• Though not all LINE elements are functional genes(~50 elements in humans), they can transpose themselves to new regions.
• Some LINE elements encode reverse transcriptase, the enzymes required for their transposition.

SINES: Short Interspersed Sequences

• SINES, such as Alu elements (common in humans), are the second most common repetitive class of repeated sequences in the human genome (~13%).
• Alu elements, measure approximately 300 nucleotides, have a poly-A tail sequence that causes them to be GC-rich.
• Alu elements are non-autonomous and require reverse transcriptase from a related group for movement.
• Alu elements are frequently implicated in various diseases and diseases involving gene regulation.

Retroviral-like Elements

• The third most abundant class of repeated DNA elements are related to retroviruses.
• These retroviral-like DNA elements, often damaged over time, lack reverse transcription abilities.
• Long terminal repeats or LTRs are a notable feature of such elements.

Satellite DNA

• Satellite DNA is associated with eukaryotic chromosomal structures, such as centromeres and telomeres.
• Centromeres have highly repetitive sequences (hundreds to thousands of repeats).
• Telomeres contain a repetitive 6-bp sequence (TTAGGG) present multiple times (~100-1000 repeats).

Repeated Genes

• Some genes are duplicated (repeated) because cells need a large amount of particular gene products, such as ribosomes, ribosomal RNAs, ribosomal proteins, tRNAs, and histones.
• Gene duplication can lead to evolution and to changes in their functions. (i.e., globin genes are derived from a common ancestor but evolved in function—like embryonic and after-birth global functions.

Ribosomal RNA Genes

• Ribosomes contain 4 types of RNA molecules: 28S, 18S, 5.8S, and 5S rRNA.
• Multiple copies of the genes encoding these rRNAs are present in the genome to support high ribosomal production.
• The rRNA genes are frequently clustered.

Genes Encoding Some Proteins

• Genes for certain proteins, such as globins, have undergone duplication events throughout evolution, with the functions of duplicate genes changing.
• The globins—specifically different types—evolved from a similar ancestor but diverged in function. (e.g., embryonic vs. adult globins)

History of Gene Duplication of Globin Loci

• Extensive duplication has occurred in globin genes.
• Genes duplicated in various places throughout evolution and have diverged in their functions—e.g., different types of globins (embryonic and adult). Examples of duplication include alpha-globin and beta-globin.

The ẞ-globin Locus

• The beta-globin locus comprises several functional beta-globin genes (expressed at different developmental stages and contain specific genes—epsilon, gamma (gy, ay), delta, and beta) and two pseudogenes (cannot code for functional proteins).

Gene Duplication Leads to Gene Families

• Gene duplication leads to the creation of gene families, resulting in regulation of tissue-specific expression and variations amongst encoded proteins.
• Gene duplication can drive speciation.

Globin Genes Are Expressed at Different Times in Development

• Different globin genes are expressed at distinct developmental stages, ensuring appropriate globin types are produced based on when in development they are needed.

How Do Genes Duplicate?

• Gene duplication occurs due to unequal crossing-over during meiosis or via retrotransposition, a method also used by transposons.

Processed Pseudogenes

• Processed pseudogenes are generated from mRNA produced during transcription.
• They lack introns and contain flanking repeats and a poly A tail.
• Transcribe to produce processed pseudogenes.

Gene Duplication and Gene Families

• Homologous genes—such as orthologs- are derived from a common ancestor.
• Paralogous genes are derived from gene duplication.

Additional Information

• Some of the original files have information that are considered to be too granular for study notes.

Explore Cot1/2 analysis, a technique used to study DNA reassociation kinetics and genome complexity. Understand how sequence repeats, fragment shearing, and reassociation temperatures impact results. Learn how Cot1/2 values relate to sequence complexity and genome characteristics.