Major Features of The Genome

Major Features of The Genome Junk DNA: Fossilized RNA Viruses (live HIV virus) destroys cells, infect and attack the immune system that can be passed on. Some insertions can be very old or can be inactive ● AKA HERVS (Human Endogenous Retrovirus ● Might affect gene expression by causing mutations ncRNAs, or translation of HERV proteins that have new functions (ex. Recombination) (can turn things on/off) ○ Binding the virus onto healthy cells ○ Fusion into one ○ Reverse Transcription: RNA to DNA from viral genome RNA ○ Integration into own DNA to make more ○ Replication then assembly then binding onto healthy cells again Repetitive Regions: over 50% of genome, transposable elements ● Doesn’t kill host, but just makes more of itself (proliferate) ex. L1 and Alu ○ Ex. L1 DNA is transcribed and translated in host genome which encodes more copies and inserts then elsewhere in the genome ● Barbara Mclintock discovered these Jumping Genes in 1950s ○ Wasn’t accepted at first due to woman but later won Nobel Prize ○ Mapped across the genome and would literally move to a different place ● Thought to be mostly parasitic, accumulated in gene-poor areas ● Can have little or no effect or can be numerous and affect many genomes functions ○ Ex. TEs can cause important mutations ○ APC is a tumor suppressor gene Non-Coding RNA (ncRNAs) ● Transcribed from genome but coding for a protein ● Can do nothing but alter the central dogma Junk DNA controversy: why sequence “useless DNA” but these non-coding regions have important functions DNA Sequencing Technologies ● Y Chromosome has frequent rearrangements (makes difficult to sequence) ● Frederick Sanger: borrowed skeleton of DNA replication to make machinery to sequence DNA ● Sanger Sequence: performing strain elongation in vitro (test tube) gel machine ○ Introduces a new sugar Dideoxyribose and dideoxynucleosides ○ DdnTPs (like a poison pill that stop chain elongation at a known nucleotide by no OH hydroxyl group binds to the next nucleotide ■ Start with a fragment (template) with a primer to add pairs AT CG ■ Add dNTPs and Polymerase to produce fragments of original fragment when the strand ends with whatever it starts with (ex. T ends to T) ■ If have DNA sequence, then can get the length &the final nucleotide ○ Things that Sped up Sanger Sequence ■ Invention of PCR: allowed segments of the genome to be easily amplified and sequenced (must know primer sequence and DNA sequence and melt it into single stranded DNA placed into water bath and some of them anneal (back together) that makes 2 molecules ● Denaturation -> Annealing -> Extension (done over and over) ● Ex. Thermal Gassier: the DNA polymerase comes from an organism that comes from here (resistant to heat) ○ Before, there were separate water baths for each stage ■ Fluorescent ddNTPs: gel machine that detects fluorescently tagged DNA separated by Electrophoresis (laser fires along nucleotides and a detection system sees what base pairs it is by its fluorescent tag) ■ Later: automation, scale, computational power of Sanger Sequencing which made it cheaper to run ● DNA sequencing uses the machinery of DNA replication ○ Sugars are part of RNA (Ribose) and DNA (Deoxyribose – removes the O in OH, structure) backbone ■ DeDeoxyribose is missing 2 Oxygens ■ Phosphate groups bind to oxygen group 3’ to 5’ (polarity) ■ One strand saved to make copy (semi-conservative replication) ■ Nucleotide: units of building helix of phosphate, sugar, base ● Floating around, bring approaite nucleotide to make it one base pair longer to make chain ■ Polymerase delivers a matching nucleotide pair (AT CG) with help of energy ATP to bind onto oxygen (hydroxyl group, forming weak hydrogen bonds ■ DNA polymerase needs a primer to start to generate double-strand and single-strand environment and nucleotides come to extend TAKE HOME MESSAGES ● Frederick Sanger: pioneer in Amino Acid (insulin), RNA and DNA sequencing ● Di-deoxy sequencing uses DNA replication machinery (chain elongation) to sequence DNA ● Di-deoxy nucleotides terminate the reaction at known nucleotides ● The polymerase Chain Reaction (PCR) allowed segments of the genome to be amplified more easily, that allowed them to be sequenced more easily ● Automation of Sanger sequencing generated exponential growth in DNA sequencing, allowing scientists to sequence the human genome