Genome Structure and Function

• The genome is an organism's complete set of genetic material, typically DNA, but RNA in some viruses.
• In eukaryotes, the term genome refers to DNA found in nuclear chromosomes.
• Genomes guide an organism's assembly and maintenance, serving as a dynamic information center for the cell.

Eukaryotic Genomes

• A eukaryotic genome equals one set of chromosomes; in humans, this means 23.
• Eukaryotes are characterized by diploidy, possessing pairs of each chromosome (e.g., 2 x 23 = 46 in humans).

Genome Analysis Approaches

• Genomes can be analyzed structurally, focusing on size and physical organization into genes and chromosomes.
• Genomes can also be analyzed functionally, to understand the number of genes, their functions, and interactions.

Revolution Through Sequencing

• DNA sequencing, emerging in the 1970s, has revolutionized molecular biology.
• DNA sequencing techniques enable reading the order of nucleotides in a fragment of DNA.

DNA Sequencing Methods and Pioneers

• The Sanger method, or chain termination method using di-deoxynucleotides, was developed by Fred Sanger.
• Fred Sanger was awarded his second Nobel prize for this effort.

Alternative Sequencing Method

• An alternative method was developed by Maxam and Gilbert at Harvard.
• Wally Gilbert shared the Nobel Prize with Sanger for their method.

The Sanger Method

• The Sanger method utilizes DNA polymerase to incorporate 2',3'-dideoxynucleotides (ddNTPs) into growing DNA chains, terminating them because ddNTPs lack a 3' OH group.

The Chain Termination Technique

• The original Sanger technique involved DNA melting to isolate complementary strands for use as a template.
• E. coli DNA polymerase I fragments were used to make copies of the template strand.
• DNA of interest was primed using a synthetic oligonucleotide.
• Four reactions were set up, each contained dATP, dCTP, dGTP, and dTTP with radiolabeling.
• Each reaction was spiked with a small amount of either ddATP, ddCTP, ddGTP, or ddTTP.
• Four reactions result in new DNA chains randomly terminated at A, G, C, or T residues by the ddNTPs.
• Each of each DNA strand has a different length based on where it has been terminated, related to the number of bases.
• These molecules are resolved using gel electrophoresis and ordered from smallest to largest to determine the DNA sequence.

Modern Sanger Sequencing Innovations

• Modern Sanger sequencing uses fluorescently labeled ddNTPs instead of radioactivity for easier detection.
• The sequencing reaction can now be assembled in one tube, enabling automated detection.
• Fine capillaries are now used to separate DNA fragments, replacing large gels.

Scale Limitations of Sanger Sequencing

• Besides the fluorescent dyes, there is a time and cost limit to using the Sanger method.

Human Genome Project

• The Human Genome Project (1990-2003) utilized Sanger sequencing; it required >10 years and cost $2.7 billion.

Next Generation Sequencing (NGS) Revolution

• DNA sequencing was revolutionized in the 2000s with Next Generation Sequencing (NGS), which offered rapid and cheaper sequencing methods.

Sequencing Throughput Comparison

• The main difference between Sanger sequencing and NGS is throughput. With NGS, millions of fragments (25-30 bases) are sequenced simultaneously per run.

Massive Parallel Sequencing

• Massive parallel sequencing (NGS) reads are computationally assembled into chromosomes and genomes.
• This is achieved through mapping reads onto the reference genome which requires storage space and power.

NGS Advantages

• NGS has become very affordable, leading to a lower cost that has lead to a delivery of a human genome overnight for under $1000.

NGS Applications

• NGS methods are being used in transformative scientific and clinical applications to study genetic variation, disease, and pathogen tracking.

Illumina Example

• The Illumina example provides a visual overview of NGS technology.

NGS Technology Implications

• One implication is the ability to catalogue human genetic variation.
• Genetic variants are pinpointed through read mapping to the Human Genome Project reference genome.

Clinical Applications

• Clinical application relates to personalized genomic healthcare. The NHS focuses on genetic variant correlation with diseases, including precision oncology, based on 13,880 tumors from the 100,000 Genomes Project.

Genomic epidemiology

• Genomic epidemiology leads to rapid identification and tracking of pathogen outbreaks. It also performs taxonomy related to sequencing.

Privacy concerns regarding genomic data

• Privacy issues have surfaced over the large databases being compiled in the genomics sector.
• For example, companies such as DNA Complete are getting personal data, raising concerns over exploitation.

Rate of Reannealing

• Information about size and complexity of DNA was gathered by studying the rate DNA reanneals.

DNA Denaturing/Melting Reversibility

• Reversible DNA denaturing/melting involves raising the temperature beyond DNA melting temperature.
• This causes the molecule to separate into strands.

Solvent factors of the DNA strands.

• The length of the DNA affectes the DNA
• So does the sequences that are included.
• Similarly, the solvent and base composition affect the reannealing too.

Rate of Reannealing

• rate in the reannealing of of DNA relates to the function is complexity itself.
• A lower temperature, and more complex DNA will lead to strands specifically pairing up in solution again.
• The rate at which sequences anneal depends on unique sequence abundance.
• Simple, homogenous DNA with repeating sequences, is less complex with a higher probability of reannealing.

Eukaryotic DNA Reannealing

• Reannealing studies of eukaryotic DNA reveal multiple phases. This is from a range of different sequence compositions which is anything from abundant, Repetitive DNA to unique sequences.

C-Value Paradox

• The C-value paradox states that there isn't connection between DNA content that correlates with organismal complexity,
• Simple organisms, like bacteria (E. coli), have a relatively low number of bases, while more complex genomes have over 3 x109 bases.

Gene Number and Density

• In Yeast, the average bases is 13 x106 while number of genes is 6000.
• In Worm C. elegans, the average bases is 1 x 108 while number of genes is 19000.
• In Humans, the average bases is 3 x109 while number of genes is 20000.
• Larger genomes does not correlate with number of corresponding genes.

Eukaryotic Genomes

• Eukaryotic genomes have unique and complex regions, and regions that are repetitive in nature.

Non-Coding DNA

• Analysis of genomes reveals that only a small fraction codes for genes and functional RNAs. For instance, human genomes, have a large amount of non coding DNA..

Non Coding Function

• Much of the human genome without a clearly defined function is referred to as junk DNA (98.5%) which is likely over estimating the amount.
• The role of non-coding DNA, is complex since there is a need for complex gene regulation.
• Certain regulatory sequences target gene expression.

Genome Footprint

• Only a small portion of the genome (25%), is considered the "footprint" of a gene, with most of the genome having unknown function.
• Functionally important non-coding RNA types, such as IncRNA and miRNA, still leaves a lot of DNA unexplained.

Repetitive DNA Elements Categories

• Tandem repeats are classified as short nucleotide stretches in head-to-tail arrangement in telomeres, centromeres, satellite DNA.
• Interspersed repeats are classified as mobile genetic elements like transposons or Alu repeats that move within it.
• Segmental duplications are classified as low-copy repeats, ranging from 1 to 400 kb, with a high sequence identity level, which typically share >90 % of the identity.

Heterochromatin

• Regions of the genome that are densely packed and contain sequence repeats for telomeres (ends of chromosomes) and centromeres.

Euchromatin

• Regions of the genome where the e genome is less packed are called Euchromatin.
• They are typically transcription genes and contain unique sequences in the chromosome arms.

Telomeres

• Unique DNA structures at the end of chromosomes

Telomere functions

• A buffer to stop genetic data from being lost.
• protective capping to protect end-to-end fusion and preserve degradation.

Telomere Sequences

• Telomeres are short DNA sequences (TTAGGG in mammals) of about 1000 base pairs.
• this sequence is attached by telomerase.
• Telomerase has is composed of a protein and RNA
• The RNA serves as a template in DNA synthesis, making it a self-templating reverse transcriptase.
• reverse transcriptases = enzyme class that uses RNA to create DNA to produce telomeres and retrotransposons.

Telomerase Activity

• It's only active during embryonic development .
• Telomerase is NOT active during somatic cell development which leads to the hay flick limit of cell division.
• Somatic cells stop dividing, since at a critical division length signals cell death.
• Does the decreasing somatic sell sizes act as a molecular clock for cellular aging?
• Cancer cells can reactivate, lengthening the hay flick unit and becoming immortable.

Telomerase Structures

• Repetitive sequence of DNA adapts to G-quadruplex (G4) structures, made of 4 GGG triplets coming together to form Hoogsteen hydrogen bonds.
• Also utilizes T loop structures where 3' end folds back & to protect telomeric structure which prevents the ends from being misidentified as damaged DNA.

Dynamic Genome Transposons

• Genome sequences change constantly as it undergoes many sequences.
• Mobile genetic elements = interspersed repeat elements
• Regions of the genome can move and insert to parts in the genome which can mutations.

Mobile agent characteristics

• Have a size/stricture and often contains protein coding chains.
• Mutations in the Doings so will depend on the source of insertion.
• Barbara McClintock found first examples in maize.

Mobile agent classification

• Class 2) use DNA transposons: use regions of DNA, that are transposed using TRANSPOSASE.
  • example is on TB3
• Class 1) use Retrotransposons: where DNA is reverse transcribed in DNA during mobilisation.
  • takes up around 42% of the genome.
  • may be active depending if proteins have autonomy
• if autonomous they can activate all proteins needed for mobilisation.
• vice vera, the effect is know as non-autonomous

Transposition overview

• Uses Conservative Transposition where excised then paste to a now side.
• Retroviruses Copy-and-paste the other region for effect.
• This DNA can move during this transposition depending in TERMINAL ends which is performed by transposon.
• LTR for long , repeats or LINE to depends.

DNA Transposons

Class 2) use DNA transposons: use regions of DNA, that are transposed using TRANSPOSASE. - example is on TB3

• Class 1) use Retrotransposons: where DNA is reverse transcribed in DNA during mobilisation.
  • takes up around 42% of the genome.
  • may be active depending if proteins have autonomy
• if autonomous they can activate all proteins needed for mobilisation.
• vice vera, the effect is know as non-autonomous

Working With DNA

manipulating DNA basics

• DNA can be easily obtained and needs manipulated in terms of cutting,past etc.
• This all leads to the understanding the function of a particular region.
• all of this is possible using different manipulations.

strand factors

• DNA must can seperate within a solution and will be renaturalised.
• This relies on temperature the length of the ds- stradns
• or the solvent. then the ionic stringth of the solevant.

DNA uses

This effects the DNA hybridization techniques are used such things as molecular bio and polymerase chain reaction ( PCR).