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Lecture 11

Lecture 11-November 16.docx

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BIOL 205
Kenton Ko

Nov 16/11 – Lecture 11 1. RNAi - RNAi is an RNA-dependent gene silencing process that used to shut down Craig Mello & Andrew Fire effects of specific genes - dsRNA is introduced into the cell where it meets the dicer enzyme that cleaves RNA, silencing them - The siRNAs recruit other components to form RISC (RNA Induced Silencing Complex) - the unwound siRNA base pairs with complementary mRNA, thus guiding the RNAi machinery to the target single-stranded mRNA resulting in the effective cleavage and subsequent degradation of the mRNA by exonucleases - this can be used to inhibit the respective gene 2. PCR - PCR uses multiple copies of a pair of short chemically synthesized primers Kary Mullis from 15-20 bases long,, each binding to a different end of the gene or region to Har Gobind Khorana be amplified - 2 primers bind to opposite DNA strands with 3’ ends pointing at each other and polymerases add bases to these primers - This process shuttles back and forth, forming an exponentially growing number of dsDNA. Requirements DNA template 1. DNA template – sample DNA that contains the target sequence and high heat applied to separate strands Primers 2. Primers – short pieces of single stranded DNA complementary to the target sequence to provide a 3’ OH dNTPs 3. dNTPs – deoxynucleotide triphosphates Buffers and salts 4. Buffers and salts Thermostable polymerase 5. Thermostable polymerase – Taq or Pfu - These polymerases were first discovered by Thomas Brock who discovered that microorganisms were growing in the boiling hot springs of Yellowstone National Park - These thermophiles can grow at temperatures between 60-108 degrees o Taq polymerase isolated from Thermis aquaticus was the first and most frequent used o Taq is stable to fluctuating temperatures Basics of PCR Cycling o Pfu (Pyrococcus furiosus) has greater fidelity Denaturation - There are 30-35 cycles comprising: Annealing 1. Denaturation (95C)- 30 s for DNA strands to separate 2. Annealing (55-60C) – 30s, the lower temperature allows primers to bind Extension to template 3. Extension (72C) – time depends on predicted product size to allow DNA to extend (30s/kb) - Increasing the cycle number above 35 has little positive effect, creating a plateau - This plateau occurs because (1) the reagents are depleted (2) the products re- anneal (3) the polymerase is damaged (4) unwanted products accumulate (e.g pyrophosphates) - PCR can amplify a usable amount of DNA (visible by gel) in ~2 hours - The template DNA need not be highly purified, a boiled bacterial colony is enough - The PCR product can be digested with restriction enzymes, sequenced or cloned - PCR can amplify a single DNA molecule (e.g. from a single sperm) - To determine if it worked, you check a sample by gel electrophoresis to see if the product was the size you expected or if there was more than one band or if the band was the right size - You may need to optimize the reaction conditions. Applications of PCR- Genetic disease - In prenatal testing, DNA is isolated from a blastocyst cell - PCR is carried out on genes using the isolated DNA as a template to produce many copies of the gene (e.g. in Thalassemia) - The PCR amplified DNA is then sequenced and compared to determine if it will cause thalassemia - This allows parents to be tested as genetic carriers and children can be tested if they’re affected by a disease. Applications of PCR – Forensics - The DNA sequences that create the variability used in this analysis contain runs of short, repeated sequences, such as GTGTGT..., which are found in various positions (loci) in the human genome. The number of repeats in each run is highly variable in the population, ranging from 4 to 40 in different individuals. A run of repeated nucleotides of this type is commonly referred to as a hypervariable microsatellite sequence, also known as a VNTR (variable number of tandem repeats) sequence - Because of the variability in these sequences, individuals will usually inherit a different variant of each VNTR locus from their mother and from their father; two unrelated individuals therefore do not usually contain the same pair of sequences - Using several VNTR loci, PCRing them and comparing to the forensic sample, you can determine which individual is the suspect. Applications –Multiplex PCR - You can even use multiple primer sets to analyze multiple genes to detect analysis deletions/duplications - Duchenne Muscular Dystrophy (DMD), is a severe degenerative muscle disorder and is the most common X-chromosome linked genetic disorder - Caused by Dystrophin (427kDa protein) located at plasma membrane of all muscle cells Q: Why is it that when an exon is - Mutations in the dystrophin gene, gross gene rearrangements are responsible missing, a band shows? for the DMD phenotype - Using multiplex PCR, 6 exons are monitored - This can also be used in phylogenetic analysis where you can determine the age of organisms from millions of years ago 3. Determining base sequence of a DNA segment - Denature 2 strands of DNA segment and create a primer for DNA synthesis Sanger/ Dideoxy sequencing that will hybridize to exactly one location on the cloned DNA segment Fred Sanger - Dideoxynucleotides lack the 3’-OH and the 2’ OH group - For DNA synthesis to take place, the DNA polymerase must catalyze a condensation reaction between 3’OH of last nucleotide and the 5’ P group of next nucleotide - Because dideoxynucleotides lack 3’ OH the reaction is stopped and synthesis is blocked at point of addition - When this is run on a gel, each ddNTP will produce bands with the 5’ nucleotide at the bottom of gel and 3’ at the top - By using a fluorescent tag for each ddNTP, you can allow the 4 reactions to take place in same test tube and undergo electrophoresis together because you’re determining sequence based on fluorescence. Nov 18/11 – Lecture 12 1. Human Genome Project - The genome was cut into random fragments by running it through a syringe Sequencing Human genome - You make a library of cloned fragments by insertion into cosmids Q: Check your understanding of this - Each clone is sequenced at least 10 times to ensure accuracy using sanger procedure method - Each sequence end is overlapped using 600bp of sequence each time to form contigs - These contigs were overlapped to form complete sequence by a computer program. Restriction fingerprinting - Large insert clones are ordered by overlapping fingerprints to create a physical map - Clones with minimal overlap are selected and divided into subclones since you can only sequence 600 at a time - Sequence subclones and assemble them to create the genome sequence. Whole genome shotgun sequencing C. Venter - The shotgun sequencing method goes straight to the job of decoding, bypassing the need for a physical map. Therefore, it is much faster - Multiple copies of the genome are randomly shredded into pieces that are 2,000 base pairs (bp) long by squeezing the DNA through a pressurized syringe. This is done a second time to generate pieces that are 10,000 bp long - Plasmid libraries are created out of 1kb, 5kb inserts - plasmid libraries are sequenced. 500 bp from each end of each fragment are decoded generating millions of sequences. Sequencing both ends of each insert is critical for the assembling the entire chromosome - Eventually you’ll have 2 clones that have sequences that lie on another clone the scaffold is determined by the vector. What has human genome sequence told us? - Less than 3% encode exons of mRNAs - We have surprisingly few genes and our genes are unevenly distributed - We have 26588 open reading frames, which are protein encoding genes. Transcripts are composed of 10 exons which are typically small and introns are large - There are many human genes that had homology/ orthologs – similar sequences - 42% of ORFs have no known functions, 38% GC, 45% repetitive sequences 2. How did genes arise - Many genes come from mRNA that was transcribed - Through a retrovirus, a DNA copy was created and this created a paralog/ duplication of original gene that creates a functional protein - Or, create a pseudogene (19 000 exists in genome) that produces no protein. - Paralogs created from mRNA lacks introns 3. Comparative Genomics - 60% of human protein coding genes have multiple splice variants, on average 3 splice variants per gene. - It’s not the number of genes that’s important but the combinations E. coli - Comparing pathogenic vs. non pathogenic strains of E.coli, O157:H7 had virulence factors, including toxins, cell invasion proteins, adherance proteins - The completed genome in Tasmanian devil has revealed how cancer can be transferred through saliva, resulting in their facial tumors - Genetics may also reveal increased susceptibility to migraine triggers 4. Next generation sequencing Pyrosequencing - "Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically - DNA is added to a single bead - This bead is placed in one of 400 000 picoliter wells - PCR amplify with polymerase, enzymes, ATP sulfurylase, luciferase - When the first dNTP is added, Polymerase catalizes its incorporation , releasing pyrophosphate in a quantity equimolar to the amount of incorporated nucleotide - ATP sulfurylase converts PPi to ATP and this reaction with luciferin generates visible light in amounts proportional to amount of ATP - Luminescence is used to detect sequence - Apyrase degrades unincorporated nucleotides and ATP before another nucleotide is added. - the method allows sequencing of a single strand of DNA by synthesizing the fixed/ immobile complementary strand along it, one base pair at a time, and detecting which base was actually added at each step Illumina/ Solexa sequencing - DNA is randomly fragmented and adapters are ligated to both ends - These single stranded fragments are attached to a surface with a dense lawn of primers - Adapters would bend and hybridize to a primer, nucleotides and enzymes are added so that double stranded bridges would be built - This would be denatured so that several million dsDNA would be generated in clusters. - All 4 labelled reversible terminators are added - Lazers are used to excite and induce fluorescence in first cycle and the image is captured with the first base of each cluster recorded - This method is repeated for the next cycle, where the 2 base is determined. The fluorescence and chain terminator are removed. - You would align data to a reference and identify sequence differences Nov/ 21/11 – Lecture 13 1. Transcription in - The 2 strands of DNA double helix separate locally and one of the separated Prokaryotes strands act as a template for RNA synthesis - Template is oriented 3’5’ and the nontemplate strand is the coding strand. - RNA polymerase positions each ribonucleotide opposite its complement base by attaching to DNA, linking aligned ribonucleotides together. - 1 RNA polymerase can synthesize multiple times on 1 gene and multiple polymerases can open transcription on a gene Promoter sequences in E.coli - The promoter controls when transcription begins and it is a conserved sequence in front of the 5’ untranslated region (UTR) - The promoter is where the RNA polymerase binds and having a common sequence allows you to use the same RNA poly several times - Promoters are found approx -35 from transcription start site - The 5’ untranslated region is the initiation site where transcription begins. Transcription initiation in prokaryotes - The RNA polymerase holoenzyme A2BB’w binds along with a specificity factor (sigma complex) at the -10 to -35 position. - The DNA is unwound and becomes single-stranded ("open") in the vicinity of the initiation site (defined as +1) and the holoenzyme complex begins dissociating, with the sigma complex coming off. - No primer is required for polymerase to bind Elongation - A transcription bubble begins to form and a ssDNA is produced, ribosomes can then jump right on - The energy for addition of a nucleotide is derived from splitting the high energy triphosphate and releasing PPi. Intrinsic Termination - There is a 40 bp sequence in 3’ UTR that ends in GC rich stretch, followed by string of 6 or more A’s which creates a transcript that forms a hairpin - This causes polymerase to pause, backtrack to encounter hairpin and disassemble. - A rho recognition site causes the polymerase to synthesize a rut (rho Rho termination utilization) site. Q: Why would prokaryotes evolve a - Rho protein binds to the rut site in RNA and moves towards 3’ end nd 2 mechanism? - When the RNA polymerase reaches the terminator, a step loop causes RNA polymerase to pause - Rho protein catches up to the open complex and separates the RNA-DNA hybrid 2. Tran
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