Molecular Biology exam 1 review.docx

**Chapter 8: Nucleotides and nucleic acid** - **Nucleotides are a monomer of DNA** **. They go in this specific order Nitrogenous base, sugar, phosphate.** - **Nucleic acid: polymer of nucleotides** **Function of Nucleotides and nucleic acid:** - **Nucleotide can be used as an ene...

**Chapter 8: Nucleotides and nucleic acid** - **Nucleotides are a monomer of DNA** **. They go in this specific order Nitrogenous base, sugar, phosphate.** - **Nucleic acid: polymer of nucleotides** **Function of Nucleotides and nucleic acid:** - **Nucleotide can be used as an energy because phosphate generates energy** - **Enzyme cofactors: Helps enzyme catalyze chemical reaction's** - **Signal transducing: changing a message within the cell** **Transmission of genetic information: DNA stores genetic information and allows it to be copied into RNA to send message into the body** **Processing of genetic information: Conversion of MRNA to protein which is done by RNA catalyst (the ribosomal RNA component)** **Protein synthesis: The processing of genetic material** **The phosphate group is the most polar and hydrophilic** **The ribose sugar is also polar (this is because oxygen is extremely electronegative)** **The nitrogenous base the most non polar so hydrophobic and it resides in the middle of the DNA (Nitrogenous bases controls base paring interactions)** **1' is the position connected to the nitrogenous base** **2' is the OH or H that's how we can tell if it's DNA or RNA. DNA has H attached at the 2'and RNA has OH attached there** **3' this connects the nucleotides together (Think 3'-5') \*there is always an OH at the 3'\*** **5' is where the phosphate group is added** **Pyrimidine and purine are distinguished by the number of their rings** **Pyrimidine has 1 ring** **Purine has 2 rings** **Telling the difference between adenine and guanine by looking at the groups they have. Adenine has an amine group attached at the top which is a NH3 group and Guanine has a carboxyl group attached which is a carbon double bonded to an oxygen** **Telling the difference between Thymine, cytosine, and uracil, (all one ring structure)** **Cytosine has an ammine group and a carboxyl group** **Thymine has 2 carboxyl group and a Methyl group** **Uracil is very similar to thymine, but it doesn't have the methyl group** **Nucleoside modifications:** - **DNA methylation: The addition or removal of a methyl group. For example, a methyl group can be added to uracil to make it thymine and can be removed from thymine to make uracil.** - **RNA Alterations: RNA can be alternated depending on the structure because structure determines function. So, the more complex a structure is the more complex the function. That's why protein is very complex because it has a complex structure.** **DNA:** - **remember that the structure of DNA has a negative charge which keeps the stands from clashing into each other** - **Antiparallel** - **Has thymine** - **5' to 3' prime** - **Double stranded** - **Very stable** **RNA:** - **Has uracil** - **Single stranded** - **Very unstable** - **Half life** **Phosphate bridges the gap between one nucleotide and the next and its called phosphodiester bond.** **Important to know that base pairing is important to help stabilize DNA however base stacking is much stronger than base pairing** **G-C bonds is stronger bonds with 3 hydrogen bonds instead of 2** **BDNA : The most structural form of DNA inside the Cell** **Transcription is protein factor that controls the expression of genes** **Replication:** - **Separating the strand then using the parent strand as a template to synthesis a new strand.** **RNA forms a hairpin loop because certain regions of the RNA strand have complementary bases (like A pairing with U, and G pairing with C). When these complementary regions come close to each other, they bond together, causing the strand to fold back on itself. This creates a loop where the non-complementary bases are, and the \"pin\" or stem is the part where the complementary bases are paired. Important to know that RNA is supposed to be single stranded** **This structure is common in RNA because it helps stabilize the molecule and can play a role in its function, such as in gene regulation or protein synthesis.** **Spontaneous RNA Hydrolysis (water being used to break a covalent bond)** - **RNA is half-life because we only need it to do what we need it for and then leave this helps control gene expression. So we only produce something when we need it ex all or DNA is the same but different organs code for different things** **DNA Denaturation** - **Breaking hydrogen bonds and weak intermolecular forces** - **DO NOT break covalent bonds** - **Base stacking also called Vander walls are stronger than the hydrogen bond that breaks first** - **Temperature melting point where 50% denatured and 50% folded** - **The higher the Tm the more stable the DNA is** - **Intermolecular bonds are called corporative bonds because they are working together to stabilize the DNA so think of the Jenga example the reason they don't all denature at once is because you're slowly breaking the bonds in the DNA and when it reaches a certain point it all comes apart ( explains why the line is sigmoidal) also that's why as the temperature increases we don't see any changes but over a short temperature range everything denatures and falls apart** - **GC bond also has a direct relationship with tm because as the GC bonds increase so does the tm** - **The reason most of DNA strand does not separate all at once is because some section that's much richer in GC bonds than AT bonds so it will separate much slower than places with AT bond.** **Mutagenesis:** - **The creation of mutation** - **It accumulates over time which is why cancer is called the disease of aging because mutations in DNA accumulates over time** - **Spontaneous reaction with H20: water and oxygen is reactive which means having water around our DNA is making it go through spontaneous events that damage it. This damage leads to mutagenesis.** - **Mutation means the changing of a nucleotide sequence** - **Some examples of environmental factors that can induce mutation is Radiation, UV light** - **So mutations isn't caused by a damaged DNA what happens is when a damages DNA isn't seen as normal our body tries to repair it and it botches the job which leads to mutation accumulating overtime** **Deamination:** - **Removal of an ammine group** - **Cytosine is usually deaminated into uracil, but the problem is uracil shouldn't be in DNA so if there is uracil in the DNA it means that cytosine has been deaminated and so then mutation occurs because DNA will then pair that uracil with an A. This is easy to spot however because we know that uracil isn't supposed to be in DNA** - **However, when 5 methylcytosine is deaminated it forms thymine, and this is a bigger problem because thymine is supposed to be in DNA so it's not easily recognizable** **Prymidine Dimers:** - **Another type of DNA damage** - **In short, pyrimidine dimers are damaged spots in DNA caused by UV light, where two thymine or cytosine bases stick together, disrupting the DNA\'s normal structure.** - **This is a problem because due to the shape DNA polymerase won't be able to replicate so the DNA then gets stuck in the nuclei and can't replicate and ends up dying** - **We use the phosphate as the energy and using the hydrolysis of those phosphate turns ATP into ADP** - **Hydrolysis of gamma (y) phosphate group is most common way to generate energy from ATP so ATP = ADP + phosphate** - **Alpha (a) + beta(B) phosphate that remove together are called phosphorylate ATP to AMP so it has more energy than just the gamma** **Nucleotides as coenzymes (chemically catalyze reactions)** - **Coenzyme a: carries two carbon units through citric acid cycle then is used to make ATP** - **Phosphate group is normal at the 5' position but in the cyclic amp it's on both 3' and 5'** - **One well-known nucleotide coenzyme is ATP (Adenosine Triphosphate). It carries and provides energy for reactions. Another example is NAD+ (Nicotinamide Adenine Dinucleotide), which helps with energy transfer in cells.** **In short, nucleotides can act as coenzymes to assist enzymes in various chemical reactions, often involving energy transfer or metabolism in cells.** **Chapter 9 DNA Technologies** **Enzymes of note** **DNA and RNA polymerases: The DNA one makes DNA and the RNA one makes RNA. Polymerase means using enzyme units to make a covalent connection** - **Reverse transcptase: using RNA as a template to make DNA** **Nucleases : cuts up nucleic acids** - **RNases and DNases: degrades RNA and degrades DNA** - **Exonuclease: binds and cuts only the 5' 3' ends because it only cuts on the outside** - **Endonucleases: cuts anywhere in the middle that's not the end** - **Restriction endonucleases: it cuts at a specific sequence of DNA** - **Ligase: glues fragments of DNA** - **Kianiaes: adds phosphate group** - **Phosphatases : removes phosphate group** **Restriction Endonuclease** - **It is palindromic so reads the same front and back** - **The arrows show us where to cut** - **Staggered arrow means that its sticky ends and strigjt down arrow means its blunt cut** **Plasmids ( cloning vector) meaning a carrier of DNA copies** - **Plasmids are small circular DNA that is extra chromosomal** - **Plasmids are really small** **The origin of replication is the most important thing on a plasmid because without this we can copy the plasmid and over time it will disappear** **The one we had in PowerPoint showed ampicillin resistance and since we know ampicillin prevents bacteria from growing we know that something being ampicillin resistance means that bacteria can grow. Resistance genes makes sure that the only bacteria growing is the one that contains our plasmid** **DNA cloning** 1. **Cloning vector is cleaved with restriction endonuclease (Form of a plasmid )** 2. **DNA fragment of interest is obtained by cleaving chromosome with a restriction endonuclease (We use sticky ends restriction enzyme to make sure that the end of our DNA matches the end of vector)** 3. **Fragments are ligated to the prepared cloning vector (DNA Ligase seals the gaps)** **Recombinant vector is DNA that has been mixed up** 4. **DNA is introduced into the host cell by typically the use of E.coli ( to get the DNA into the cell there are multiple ways either poke holes using salt and allow the DNA to diffuse in or using an electrical current and allowing the DNA to flow. Getting the DNA into the cell is called transformation** 5. **Cloning of transformed cell produces many copies of recombinant DNA** - **Doesn't require restriction endonuclease to make a cut** - **Start off with linear DNA** - **Uses exonuclease to make regions that's complementary to each other** **Artificial chromosomes (BAC) Bacteria making a giant piece of DNA** - **Cloning sites within lac Z (codes for protein betaglucosidase)** - **Par genes are important because without it there's no guarantee that a chromosome will separate** - **Betaglucosidase gives color to X gal changing it from colorless to blue** - **So if we put the DNA of intrest into the lac z gene it would disrupt it meaning betagaltosidase would not be blue which is why the outcome that's white is what contains the gene of interest** **Prpkaryotes** - **Circular chromosomes** **Eukaroytes** - **Linear chromosomes** - **Much larger** - **Have more origins of replication** - **Yeast are eukaryotes** **Artificial chromosomes (YAC)** - **Instead of using the PAR gene like we do in bacteria we use centromeres that pulls the chromosomes apart** - **YAC: starts off circular then becomes linear because its easier to clone the plasmid in bacteria which is circular** **Expression Vectors (expressing genes)** - **Promoter region** - **Repressor inhibits gene expression** - **Operator region** - **Ribosome binding site** - **Transcription terminator sequence** - **Helps regulate and control gene expression** - **Open reading frame (start codon to stop codon)** - **utilize the lac operator** **PCR (polymerase chain reaction) this is copying one specific piece of DNA over and over again** 1. **Denaturation: using heat to separate DNA by using a machine called thermocycler** 2. **Add synthetic oligo-nucleotide primers so annealing. We put together primers that anneals to template sequence because DNA Polymerase can only work by adding on to existing nucleic acid.** 3. **Add taq (thermostable) DNA to catalyze 5'- 3' DNA synthesis. So extension; we are extending the primer using the template sequence as a base to copy from we copy from the DNA template because it is complementary** **PCR is exponential amplification** **Variations of pcr** - **Reverse transcriptase PCR: using RNA as a template to make DNA. It converts MRNA to CDNA (no introns) with reverse transcriptase enzyme** - **Quantitative PCR or real time PCR: Measure the amour of DNA in a sample in real time. What we use for covid testing. In this PCR reaction we include a dye as the PCR cycle goes up the signal increases in a sigmoid fashion** - **Directed mutagenesis: specific mutation using PCR and primers that are not complementary. The mutation is put in those primers** - **DNA fingerprinting: we amplify multiple regions of specific parts of genome called STR. Short Tandon repeats.** **Labrtory analysis of DNA** - **Gel electrophoresis : separate molecules based on their movement we use charge. We know DNA is negatively charged so it moves down through the gel.** **A lot of genes are useful when it comes to treating diseases for example insulin we can make it now using proteins as opposed to using animals.** - **Protein are used medicaniry** - **Antibodies: they tool blood from patients who survived a diseases to give to someone ppl who are fighting that disease called convalescent antibodies** - **Growth factors stimulate growth of specific cells** - **Vaccines: most of them today are protein based. It takes a long time to make them six months to be exact so if we had a quicker way to develop the vaccine we could do a better job at vaccinating** **Purification of recombinant proteins** - **Proteins are purified using physical properties such as charge affinity (tendency to bind to things) and size** - **Proteins can also be tagged by recombinantly binding it to another protein** - **Pufrication of protein is important because we want to be able to isolate a single protein from everything else. We wouldn't want to give patients anything other than the protein, so we don't trigger any reaction** - **We use chromatography to purify protein and there are two stages; stationary which includes what protein binds with and mobile; typically, the phase protein is in** 1. **We have the target protein** 2. **We then fuse another protein into the gene for origin protein** 3. **So then its covalently attached to another protein we can then use to purify** **Colom chromatography** **Cylinder is column and the protein is put in from the top and flowed down. The thing with affinity for the stuff the column is made of will stick to it and anything without will pass through. Then elusion happens and that's when it leaves. The protein comes out and we have a pure sample of protein** **Another interesting way to use fusion protein is tagging them to a reporter gene for example GFP(it floruses when seen underneath a UV light)** **Transgenic organisms and gene therapy** - **Can use recombinant DNA technology to directly modify genome of organisims** 1. **Electroporation: using electric current to get into the cells** 2. **Microinjection: a needle that pokes a hole into the cell and allows you to inject DNA into the cell without destroying the cell** 3. **Liposomes: lipid nanoparticle so we put MRNA into lipid particle and then the lipid particle fuses to the cell and dumps its cargo into the cell** 4. **Viral vectors: virus are good at getting nucleic acid into the cells we use virus that cant replicate in the host** **Sequence human genome** - **Price has gone down significantly** - **Knows the whole genomic sequence helps plan for what diseases someone can get and helps us see what lifestyle choices would be better ''** **Composition of the human genome** - **Only a small fraction of our genome codes for protein** - **A lot of our gene is introns (not expressed)** **Accelerated evolution in regulatory DNA** - **Humans ability is much more enhanced than other mammals** **SNPs and Haplotypes** - **SNPs are individual nucleotide (single nucleotide polymorphism)** - **Most human DNA is the same** - **Haplotype are unique combination of SNPs that always occur together it can be used to distinguished different individual and disease states** **Nucleic acid sequencing** **Sanger Sequencing** - **Cant sequence millions at the same time** - **Uses a variation of PCR** - **Sequencing by synthesis** - **Old school** - **Terminator nucleotide (doesn't have an OH at the 3' end so it blocks addition of new nucleotide and stops the pcr process** - **We use fluoresces labeled terminator so we can see it** - **First, DNA is copied, but special \"terminator\" nucleotides are mixed in(unreversible). These terminator nucleotides are like regular nucleotides (A, T, C, G), but they have a twist: when they\'re added, the DNA chain stops growing.** - **Each type of terminator nucleotide is labeled with a different fluorescent dye, so when a chain stops at one of these nucleotides, you know exactly which one it is.** - **Fluorescent dye:** - **The dye attached to each terminator glows in a specific color when exposed to a laser. For example, if a terminator A is added, it glows in one color, C in another color, and so on.** - **This makes it easy to \"read\" the DNA sequence. As the laser scans the DNA fragments, the different colors reveal the order of nucleotides, one after the other.** - **All the DNA fragments (which stop at different points due to the terminators) are separated by size. The smallest pieces come out first, followed by longer ones.** - **By reading the colors from shortest to longest, you can figure out the exact DNA sequence.** **Reversable terminator sequence** - **Also sequencing by synthesis** - **Also uses fluorenes labeled terminal nucleotide** - **Can run millions of DNA at the same time** - **Indirect method** - **In this process, the DNA is copied one letter (nucleotide) at a time, using special \"reversible terminator nucleotides.\"** - **These nucleotides are similar to the ones in Sanger sequencing because they stop the DNA from growing after being added, but the key difference is that the stop signal can be reversed.** - **Like in Sanger sequencing, each of these special nucleotides (A, T, C, G) is labeled with a different fluorescent dye. So, when a nucleotide is added to the DNA strand, it glows in a specific color, telling you which one was added (A, T, C, or G).For example, an \"A\" might glow red, \"C\" might glow green, and so on** - **After the glowing nucleotide is detected and recorded, the \"terminator\" part (the thing that stopped DNA growth) is removed or reversed. This allows the DNA strand to keep growing.** - **Once the terminator is removed, the next nucleotide is added, and the process repeats: the new nucleotide is detected by its fluorescent color, recorded, and then the terminator is removed again.** **Nanopore sequencing** - **Not sequencing by synthesis** - **Uses electrical current** - **Uses helicase to unwind the DNA** - **Direct method** - **Sequences millions of DNA at the same time** - **it reads DNA directly as it passes through a tiny hole (a nanopore)** - **The changes in the electrical current are different for each nucleotide (A, T, C, or G).** - **By detecting these changes in the current, the sequencing machine can figure out the order of the nucleotides in the DNA strand.** - **Nanopore sequencing doesn't require copying or stopping DNA synthesis like Sanger or reversible terminator methods. It reads the DNA directly as it passes through the nanopore.** - **DNA inside of a cell** - **DNA is located mostly in the nucleus** **Looking at E.coli it's a prokaryote so it doesn't have a nucleus so it compacts its DNA into a nucleoid.** **DNA super coiling** - **Compacts DNA** - **Think of the phone cord where it's locked at both end but coiled up in the middle ex. Since plasmid is circular and there's no end it warps around itself** - **As we increase supercoil DNA becomes more compact** - **Supercoil is dependent on something called the linking number which is basically the amount of time one strand warps around the other** - **When it comes to DNA the number of times a double helix warps around each other depends on how many nucleotides it takes to get a 360 turn** - **When a LK is undefined it's because there is a break in the DNA** - **When LK is at 200 it means that its fully relaxed and there's no supercoiling** - **There is positive LK and negative LK** - **Lk -2 is removing links and LK +2 is adding links the problem with them having the same number but different sign is that they look exactly the same it's just they are warped in different directions** **Relaxed DNA** - **Takes more space** - **Runs slowly** **Highly coiled DNA** - **DNA runs faster** **Topoisomers ( remember that isomers have the same formula they are just drawn differently)** - **What it does is cuts the DNA and either wind or unwind** - **Important to know that super coiling can only be changed by cutting somewhere** **Type 1** **Type 2** - **They make double stranded breaks** - **Adding or removing 2 links to the DNA +2 or -2** **Topoisomerase Inhibitors** - **Drugs for treating infections and cancer** - **Ciprofloxacin: (antibiotics that is used for bacteria infection. Binds bacteria to topoisomerase** - **Doxorubicin: chemotherapy drug that blocks the proliferation of tumor** **Most DNA is sightly negative supercoiled. So can open the DNA and it can unwind easily** **Nucleolus: little nucleus** **Chromatin: mixture of DNA with protein it binds to** **Heterochromatin** - **Tightly compacted DNA** - **Darker stain** - **Less gene expression happening** **Euchromatin** - **Loosely compacted DNA** - **Lighter stain** - **More gene expression** **As we remember a huge part of our DNA does not code for anything. A lot of DNA is super tightly compacted because it is never going to be expressed** **Centromeres: an epigenetic property** - **Histone is a protein associated with DNA that tells us where a centromere should be** - **DNA warps around the Histone** - **Histones have 8 different subunit and technically 2 copies of each subunit**