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Why sequence DNA?
- Gene finding, systematically search through nucleotides for open reading frames
- Insights into evolution: Phylogenetic trees
- Identification of functional sequences such as enhancer, promoters
- Finding mutations
- Biotech and engineering, such as GMO and agriculture
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Explain gel electrophoresis
- We use an agarose gel with wells filled with DNA solutions
- Voltage is applied across the gel.
- DNA is negatively changed, so it migrates through the gel towards the positively charged electrode.
- Long molecules migrate slower.
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Explain Maxam-Gilbert sequencing
- Prepare homogenous ssDNA
- Add P-32 isotope as 5’ phosphate
- Cleave at specific nucleotides, run a G reaction, A reaction, T reaction, C reaction
- Run fragments on gel
- Detect fragments using P-32
- Fragmentation patterns across cleavage reactions indicate the sequence of the starting DNA
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Explain DNA replication
- Requires a template strand.
- Requires a primer
- Polymerase adds dNTP to the 3’ end of the primer, pairing with the nucleotide across the template strand.
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What do we need for Sanger sequencing.
- DNA template
- Primer (end-labeled with p-32)
- dNTPs (all four standard nucleotides)
- Single ddNTP (at low concentration)
- Polymerase, buffer
- Chain terminating nucleotides don’t have the 3’ hydroxyl, which blocks extension by polymerization. They can also be fluorescently marked.
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Explain old Sanger sequencing.
- Perform reaction with each ddNTP separately.
- Run soup of elongated DNA molecules on a gel.
- Each fragment indicates where a ddNTP was incorporated.
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Explain modern Sanger sequencing
- Uses chain-terminating ddNTPs with fluorescent labels.
- All 4 ddNTPs included, but at much lower concentration.
- Dye-labeled segments are applied to a capillary gel and subjected to electrophoresis.
- Laser and detector excite fluorescent label and detect it.
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Explain next generation high-throughput sequencing
- A suite of alternative (to Sanger methods) sequencing technologies. These are the core technologies for genomics and personalized & precision medicine.
- Illumina sequencing dominates - many orders of magnitude cheaper and faster than traditional Sanger sequencing.
- Nanopore & PacBio: Single-molecule real-time sequencing & others
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Explain Illumina sequencing
- DNA molecules are covalently attached to a glass slide.
- Uses chain-terminating nucleotides that are fluorescent with different colors. No normal nucleotides.
- We incorporate fluorescent, chain-terminating nucleotide.
- After incorporation, ee wash away unincorporated nucleotides, record the color.
- We then cleave the blocking group and fluorescent label and replace with hydroxyl group. This allows for us to continue to build on the same DNA molecule.
- We repeat this process.
- Many DNA molecules are sequenced in parallel (a billion dots in a glass slide).
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What is recombinant DNA
- Also called chimeric DNA
- A new DNA sequence created by combining existing sequences.
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What are restriction endonucleases
- Enzymes that recognize specific sequences in DNA, and cleave the DNA within the recognition sequence.
- Large variety of restriction enzymes with three large sets: blunt ends, 5’ overhang, 3; overhang.
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Explain biology of restriction endonucleases
- Bacteria possess many pathways to transfer DNA between cells/species
- Restriction endonucleases perform a protective role by digesting foreign DNA - the foreign DNA is restricted
- Host DNA is protected from cleavage by site-specific DNA methylases, methylation of the recognition sequence interferes wit function of the restriction endonucleases - the host DNA is modified
- Pair of enzymes: a methylase and a restriction endonuclease, which have overlapping recognition sequences
- Methylation of DNA is a recurring theme across biology - half of mammalian CpG dinucleotides are C methylated.
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DNA ligase
E.coli or T4 ligase will ligate nicks with 3’OH and 5’P
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What are cloning vectors
- Contain DNA of interest; allow for replication and purification from E.coli.
- Plasmids have
- Origins of replication
- Drug resistance marker
- Multiple cloning site (series of recognition sequences for different endonucleases stitched together)
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How do we use restriction enzymes to make recombinant DNA
- We digest DNA with a specific RE.
- We insert DNA with matching overhangs.
- RE will be covalently attached after addition of DNA ligase, which joins the phosphate backbone’s 3’ OH and 5’ P.
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Explain Polymerase Chain Reaction
- The polymerase chain reaction (PCR) allows researchers to amplify a target region from a genome.
- Two oligonucleotides or primers are used to amplify a target region of DNA. The researcher designs these primers based on knowledge of the target region they wish to amplify. One primer is complementary to one strand of the DNA at one end of the region to be amplified and the other primer is complementary to the other strand of DNA at the other end of the region.
- The size of the region to be amplified is defined by the 5’ ends of the two primers by DNA polymerase from thermophilic bacteria.
- The final PCR product extends from the 5’ end of one primer to the 5’ end of the other primer. The 3’ ends of the two primers must point towards each other.
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What do PCR reactions require?
- Template
- Two primers
- A heat-stable DNA polymerase
- A supply of 4 dNTPs
- Buffer
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Explain each PCR cycle
- Each PCR cycle consists of the following 3 steps:
- Denaturation: The first step is at high temperature (98C) to melt the double stranded template into single strands
- Annealing: The temperature is lowered to 56C (depending on the primers) to promote annealing.
- Extension: The temperature is raised to the working temperature of the polymerase (72C).
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Explain the differences between RNA and DNA and their consequences
- DNA is usually double stranded, and therefore constrained in structure. RNA is single stranded, and thus less constrained and inherently unstable.
- The 2’ hydroxyl group on ribose - additional hydrogen bonding and non-covalent interactions are possible.
- Hydroxyl ion (base) can attack the 2’ hydroxyl of RNA, which can cause RNA to self-cleave by attacking the phosphate connected to the 3’ carbon. (H gidiyor, P’ye baglaniyor, o da bir sonraki CH2den ayriliyor)
- Thymine replaced by uracil allows for wobble pairing. (G-U wobble pair)
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Why isn’t RNA used as genetic material?
- Unlike DNA, RNA is not stable (hydrolysis) in aqueous solution.
- No organism (animal, plant, bacteria etc. uses RNA as its genetic material)
- Many viruses use RNA as their genetic material, including those that infect animals, plants, and bacteria.
- RNA is short, and environments are not too basic. This makes hydrolysis slow and minimizes its effect.
- All viruses are dependent on RNA-dependent RNA polymerase.
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What are the three groups of viruses that use RNA as their genetic material?
- Positive strand (polio, cold, Zika, West nile, SARS) Can act as both the genome and the mRNA. Many viruses carry RNA polymerase in their protein.
- Negative strand (ebola, measles, rabies, influenza). Viral RdRP is one of the first proteins produced by these viruses.
- Double stranded: rotavirus
- All viruses are dependent on RNA-dependent RNA polymerase.
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Explain retroviruses
- Using RNA to make DNA
- Retrovirus merges with the host cell. RNA genome enters the cytoplasm of the host cell.
- Reverse transcriptase makes first a single stranded, then double-stranded DNA from the viral RNA. This is an exception to the central dogma.
- The double stranded DNA then goes into the nucleus and incorporates into the genome.
- Viral genes are transcribed.
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How can we use reverse transcriptase?
- Used when we want to make a piece of DNA that only has sequences in the mRNA, as opposed to the genome. Key uses include RT-PCR, recombinant DNA technology.
- mRNA template is annealed to a synthetic oligonucleotide dT primer which recognizes the poly(A) tail.
- Reverse transcriptase and dNTPs yield a complementary DNA strand. (cDNA)
- mRNA is degraded with alkali.
- cDNA is amplified with PCR
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Typical RNA structures
- The molecule looks single stranded, but is stabilized by structural secondary motifs such as bulges, internal loops and hairpins.
- Tetraloop: Series of non-covalent interactions that further stabilize the loop region of RNA.
- Pseudoknots are made from base-pairing in distal regions.
- Kissing loops are base-pairing between loops of discrete bases.
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RNA structural elements not present in DNA
- Pseudoknot - distal regions of complementary RNA within the same molecule that forms regions of double-stranded RNA via base-pairing
- Kissing loops - regions of structured RNA that form loops where the nucleotides on the end of the loop base-pair with nucleotides on the end of another loop
- Single-stranded regions - regions of a structured RNA in which there is no base pairing, exposing single-stranded RNA
- Tetraloops - a region at the end of an RNA loop in which four nucleotides form a stable structure via a combination of base-pairing and base-stacking.
- Hairpin - a double-stranded region of RNA with a loop where the molecule bends back and base-pairs with itself
- Wobble pairing - base-pairing between bases other than A-T/U and G-C, due to the presence of Uracil instead of Thymine and a hydroxyl on the 2’ carbon of the nucleotides
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Explain catalytic activity of RNA
- Protein: Form specific and very precise catalytic cores.
- Some very unusual RNA molecules do the same thing. Hence, some RNAs can function as enzymes.
- An example includes ribosomal RNA, which catalyzes translation.
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Explain 3 exceptions to the central dogma
- Reverse transcription (RNA to DNA)
- RNA replication with RNA polymerase
- Ribosomal RNA has a protein-like catalytic function.
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