Unit 9
ion semiconductor sequencing (Ion Torrent)
-4 dNTPs introduced one by one in repeating sequence -addition at a certain spot detected by measuring protons released
What is needed in order to amplify a segment of DNA using PCR?
-DNA -free dNTPs -primers (in abundance) -DNA poly that can withstand high temps -buffer
describe the synthesis of cDNA molecules
-anneal DNA oligonucleotide primer to mRNA -reverse transcriptase yields complementary DNA (cDNA) -RNA degraded with alkalai -known oligonucleotide sequence ligated to cDNA -primers complementary to known sequence annealed -DNA poly and dNTPs extend primer and yield double stranded cDNA
describe the photolithographic method for fabricating DNA microarrays.
-computer programmed with desired sequences -Nucleotide groups rendered inactive by photoliable blocking groups -opaque screen covers areas where addition not desired, flash light to activate where addition desired -wash with solution of nucleotide you want to add -repeat to create full sequences
describe a procedure used to tag proteins thereby facilitating their purification using affinity chromatography techniques.
-fuse gene encoding target protein to gene for tag protein that binds simple ligand with high affinity -produces fusion protein -run through affinity column with ligand, tag protein will adhere -elute with free ligand or high salt conc. Example: GST tag protein binds Glutathione
Discuss how fusions with the gene for GFP can be used to localize specific proteins within cells and organisms
-fuse target protein gene and GFP gene -gives fusion protein with GFP fused to target -target protein is thus flourescent, and can observe its movement and location in a living cell
Discuss how CRISPR / Cas 9 can be used to "knock out" a gene.
-introduce CRISPR/Cas9 plasmid -produces complex with endonucleases and sgRNA -scans DNA for matching sequence -if both endonucleases active, makes double stranded cut -nonhomologous end joining leads to gene mutation and knockout
Discuss how CRISPR / Cas 9 can be used to make a specific change in a gene sequence.
-introduce CRISPR/Cas9 plasmid, and recombinant fragment -produces complex with endonucleases and sgRNA -scans DNA for matching sequence -if only one endonuclease active, makes single stranded cut - recombination with added fragment leads to homology-directed repair, recombinant fragment introduced in desired location
Discuss how a DNA microarray (or chip) can be used to study changes in gene expression that accompany development.
-isolate mRNA from cells at different stages -use reverse transcriptase to make cDNA using flourescent nucleotides -allow cDNA to anneal to microarray -color shows where genes being expressed -different colors show where genes being expressed differently -mixed color shows no change in expression -gradient of color can show level of expression
Describe the process of annealing and joining recombinant DNA
-need the same endonuclease cut both fragments (ideally making sticky ends) so they will be complementary and anneal -use DNA ligase to form phosphodiester bonds to covalently join the fragments
Discuss how plasmids are used to clone DNA.
-plasmid cleaved -desired DNA cleaved and ligated to plasmids -plasmids transformed into host system -grow and use product of selectable marker to determine which uptook plasmids using 2 markers: insertion of desired DNA knocks out one of the genes -select for cells that uptook plasmid by growing on plate where only cells with (intact) marker gene present -move colonies into grids on two plates, one on which the plasmids with the gene knocked out from recombination will die to determine which colonies not only have plasmid, but have the recombinant plasmid
describe next-generation reversible terminator sequencing (Ilumina)
-primer complementary to known oligonucleotide added -add blocked, flourescently labeled nucleotides -nucleotide that adds flouresces, reveals identity of addition to each cluster -wash -remove labels and blocking groups -wash -add new, blocked labeled nucleotides
describe the general preparation for next-generation
-sheer genomic DNA at random locations, generates fragments a few hundred bp long -ligate known oligonucleotides to each end (provides reference point) -immobilize fragments on solid surface -amplify with PCR, creates cluster of identical fragments -screen clusters
single-molecule real-time (SMRT) sequencing
-single polymerase immobilized at bottom of each engineered pore on flow cell -polymerase captures genomic segments as they diffuse into pore -labeled dNTPs diffuse in, each addition releases colored flourescent group -light detection system records color of reulting light flash from each pore
Problem 5 (pg. 357): outline a PCR-based test for HD that could be carried out using a blood sample. Assume the PCR primer must be 25 nucleotides long.
-synthesize primer for the 25 nucelotides upstream of the repeating region -run PCR -run product through gel -there will be results of varying length for different patients. Shorter length indicates healthy, longer indicates HD
Describe how PCR could be used to detect HIV infection at an early stage, including what information about HIV would be needed to begin with.
-take cell sample from patient -collect DNA for PCR -make primers for known region of virus DNA -run PCR to amplify all DNA -run product through electrophoresis gel -if virus is present, a characteristic band will appear (viral DNA usually much shorter than eukaryotic)
Problem 13 (pg. 314): One strand of a chromosomal DNA sequence is shown below. An investigator wants to amplify and isolate a DNA fragment defined by the segment shown in red, using the polymerase chain reaction (PCR). Design two PCR primers, each 20 nucleotides long, that can be used to amplify this DNA segment. The final PCR product generated with your primers should include no sequences outside the segment in red.
-write complementary strand for first and last 20 nucleotides -sequences at 5' ends are primers for 3' ends, synthesize those
The bands seen on the electrophoresis gel differ in length by how many nucleotides?
1
describe how fusions with epitope tags can be used to identify interacting proteins.
1) epitope tags used for immunoprecipitation -make fusion protein with target and epitope -express and make crude extract -introduce antibody, binds and precipitates -collect precipitate -run in gel, interacting proteins will give bands in addition the the band of the target protein 2) Tandem affinity purification (TAD) tags -fuse target with 2 tags that bind ligands -run through column for 1st tag (retains target with all interacting proteins) -elute and cleave first tag -run through 2nd column for 2nd tag (retains target with interacting proteins with high affinity, less false positives) -elute
Describe the five general procedures in DNA cloning
1. obtaining DNA to be cloned: need endonuclease and cut sites around the gene of interest 2. selecting small molecule of DNA capable of autonomous replication (cloning vector, usually modified DNA molecules found in bacteria, yeast, viruses) 3. Joining the 2 DNA fragments covalently: DNA ligase links the cloning vector to the desired DNA sequence, product is recombinant DNA 4. Moving recombinant DNA into host organism (want to use host to replicate): transformation, etc. 5. Identifying host cells that contain recombinant DNA: vector usually has selectable property
Why do plasmids always contain each of the following regions: 1) the selectable marker gene (ex. antibiotic resistance gene) 2) the origin of replication (ori) 3) unique recognition sequences for restriction endonucleases
1. so that you can identify which cells/colonies took up DNA (can also use reporter genes, but less common) 2. so that the cloning vector can be replicated and propogated 3. so that the plasmids are targets for the specific restriction endnuclease you want, provide site for interstion of DNA
What fraction of the genome encodes protein (protein coding genes)?
1.5%
Problem 17 (p. 315): Predict the gel for each reaction 1: uses ddTTP 2: uses ddGTP 3: no dTTP, only ddTTP 4: no ddNTPs
1: bands at each T location 2: bands at each G location 3: one band at the bottom (can only add one nucleotide) 4: one band at the top (full synthesis only)
How many protein coding genes in the human genome?
20,000
For each copy of the target sequence that you started with, how many copies do you expect after cycle 10 (assuming that the amplification performs perfectly in each cycle)?
2^10
What fraction of the human genome consists of genes (protein coding genes plus introns)?
30%
how many cycles are usually run in PCR?
32
How many reactions are included in a typical Sanger DNA sequencing experiment?Describe the composition of each reaction.
4 Each one uses only one base for the ddNTP, so each reaction gives the locations of that base
Explain how RNAi can be used as a gene silencing method.
= RNA interference -create duplex RNA complementary to mRNA you want to silence -introduce via a vector -RNA product in host cleaved by Dicer into short segments = siRNA -bind to mRNA and silence them
What does qPCR stand for and why is it used?Describe how qPCR works.
= quantitative PCR used to estimate realtive copy numbers of a DNA sequence in a sample (to reveal amplification of a certain gene) uses probes that have a flourophore and a quenching protein. when alone base pairs to self, quenching protein next to flourophore, no flourescence. When complementary DNA present, anneals to DNA, flourophore not blocked, flouresces. set threshold for flourescence level, measure as increases (more DNA for probes to anneal to). those that cross faster had more DNA present initially.
What does RT-PCR stand for and why is it used?
= reverse transcriptase PCR used either for systems that only use RNA, or to observe gene expression (by looking at RNA products) first step of PCR uses reverse trascriptase to produce cDNA, can then proceed with regular PCR
What is a DNA library? Name and describe two types of DNA libraries
DNA library is a collection of DNA clones genomic library = complete genome of an organism is cleaved into thousands of fragments and cloned in cloning vectors cDNA library: only contains DNA that are expressed (mRNA -> cDNA via reverse transcriptase, then insrted in vectors and cloned)
What is a DNA Microarray?
DNA segments from genes of known sequence synthesized on solid surface via photolithography -contains many thousands of spots containing sequences from a particular gene
Give an example of a restriction endonuclease that generates sticky ends and one that generates blunt ends.
EcoRI gives sticky ends Pvull gives blunt ends
What is a His tag and what is it used for?
His tag is 6 His residues added to make a fusion protein, coordinate with high affinity to Ni2+ in affinity column
A normal enzyme is denatured above 50-60°C, but in each cycle of PCR the mixture is heated above 90°C. Explain why the use of DNA polymerase from the bacterium Thermus aquaticus (found in hot springs) made a big improvement in this procedure
It does not denature during the heating process, so you can separate the DNA and anneal primers without interrupting the function of DNA poly
Given the sequence and primer, write the sequences that would be produced using the dideoxy analog of dATP. 3' AGCTATTCGTGAC 5' 5' TCGA 3'
TCGATA TCGATAA TCGATAAGCA
Thousands of restriction endonucleases have been discovered. Are restriction enzymes made by eukaryotes or prokaryotes? What is their function in vivo?
They are found in bacteria, where they function to recognize and cleave foreign DNA
Suppose you want to use oligonucleotide directed mutagenesis to make a mutant gene that encodes a protein containing phenylalanine rather than tyrosine. Given the following DNA and protein sequence, write the sequence of the primers you would design to make the mutant protein. Cys Ser Leu Tyr14 Gln Leu Glu TGT AGT CTC TAT CAA TTA GAA (coding strand)
Tyr is coded by UAU, Phe coded by UUU use primer that replaces A with T in that position
What fraction of the human genome do transposons represent?
a little less than half (~47%)
describe next-generation pyrosequencing (454)
addition of nucleotides detected by flashes of light -pulse dNTP one N at a time across surface in repeating sequence, retain long enough to be incorporated, then destroy -successful addition produces pyrophospate byproduct -sulfurylase transforms into ATP -luciferase catalyzes light emitting reaction when ATP present => flashes only occur in clusters where the nucleotide was added, and intensity signifies how many were added => gives the sequence of each cluster
Restriction Endonucleases
an enzyme that cuts DNA at specific sites, producing small fragments used in genetic engineering.
What is the biological function of CRISPR / Cas in bacterial cells?
and adaptive defense system for prokaryotes agains viral infections. Used to recognize and cleave invading viral DNA
Discuss the restriction-modification system
any sequences in the host DNA that would be recognized by restriction endonuclease protected via methylatation by a specific DNA methylase
describe how DNA primers and probes of a specific sequence are synthesized
attach strand to silica, use blocked nucleotides. -remove protecting group, wash with desired nucleotide repeat oxidize and remove all protecting groups for final product
Why is it possible with this method to have only a single reaction mixture?
because the added nucleotides are distinguisable. When you analyze the gel and run it past a laser, you can see the color for each band, and know which nucleotide was added.
describe GFP and its variants.
beta barrel protein, flouresces -Tyr, Ser, Gly residues rearrange and undergo oxidation, gives flouresence protein engineering can give different colored variants
What are sticky and blunt ends?
blunt ends: endonuclease cuts at the same spot on both strands, no overhanging nucleotides sticky ends: endonuclease cuts at different spots on the two strands, one is longer than the other
After separating the fragments, how do you know which fragment stopped at which residue?
by exciting them with a laser and detecting the color of the flourescence emitted
What does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
Explain how the short sequences generated using this method are assembled into contigs?
computer aligns overlapping fragments into continous sequence = contigs
discuss the Sanger method for DNA sequencing.
dideoxy chain termination sequencing need: template DNA, radioactive primer, dNTP, ddNTP, polymerase -ddNTPs terminate synthesis, end up with different length chains (added in low concentration so only stops sometimes, gives full range of possibilities), produces a ladder in the gel that indicates where each base is located (in gel, shorter travels farther so bottom is 5', top is 3' end)
Protein-protein interactions can help.....
elucidate protein function
Why is it necessary to have a promoter in the expression vector when using cDNA inserts?
eukaryotic transcription sequences will not be recognized by prokaryotic machinery, need bacterial promoter to transcribe gene
Why does this method allow more sequence information to be derived from a single sequencing reaction?
faster, so can sequence very long molecules fairly quickly, much more efficient
Describe the automatable version of Sanger sequencing
flourescent tags attached to the dideoxynucleotides, one reaction run
discuss how indirect immunofluorescence microscopy can be used to determine the sub-cellular localization of a specific protein
if GFP not ideal (changes folding and function, fusion protein inactive, not expressed highly enough to be seen) can use immunoflourescence microscopy -death and fixation of cell -wash with primary antibody that binds to target protein -wash with secondary antibody that binds primary antibody, has flourescent group -vizualize flourescence under microscope, can see location of protein
Fusion proteins and immunofluorescence can be used to......
localize proteins in cells
How could you use a microarray for medical diagnosis?
make microarray with DNA genes known to be linked to disease. make cDNA from expressed products, anneal to microarray, color shows if diesease linked genes are being expressed.
Discuss why is it desirable to have a regulatable promoter in an expression vector.
overproduction can kill the cell
PCR
polymerase chain reaction used to make many copies of a specific segment of DNA
Discuss the importance of each region of the plasmid: promoter, operator, ribosome binding site, polylinker sites, termination sequence, selectable marker gene, ori, repressor gene
promoter: eukaryotic transcription sequences will not be recognized by prokaryotic machinery, need bacterial promoter to transcribe gene operator: want a good promoter, but if unregulated can over produce and kill cell, need way to regulate RBS: provides sequence signals for efficient translation of desired mRNA polylinker sites: cut sites for endnuclease, where you can insert DNA termination seq: improves amount and stability of mRNA selectable marker: allows for selection of cells with plasmid ori: allows for propogation of plasmid repressor gene: creates repressor protein to regulate trascription
How is radioactivity incorporated into the sequences?
radioactive isomers are used to construct the primers
Distinguish between radioactivity and fluorescence.
radioactive isotopes emit ionizing radiation (detect radiation, single detection), flourescent groups emit light when excited (detect light, many possibilities)
What are transposons?
segments of DNA that can move from one location to another in the genome, genetic parasites
What are micro-RNAs?
short noncoding RNAs used in gene regulation, complementary to regions of mRNA, promote degradation and suppress translation
Define SNP
single nucleotide polymorphism single base variations, gives some of the differences between individuals and organisms
plasmid
small circular DNA molecule, replicates separately from host chromosome can undergo horizontal gene transfer
show how primers can be used to create restriction endonuclease cleavage sites on your PCR product to facilitate cloning.
synthesize a primer where the end has a cleavage site. This part won't anneal but will be incorporated into the new DNA. This product DNA can thus be cut by restriction endonucleases.
distinguish oligonucleotide directed mutagenesis from the synthetic insert method
synthetic insert mutagenesis: cleaves recombinant DNA with restriction endonuclease, inserts sythetic DNA segment with mutation (use when have suitable restriction sites around area you want to modify) oligonucleotide directed: plasmid denatured, oligonucleotide primers with single mutation annealed to single strands. DNA polymerase extends and incorporates mutagenic primers into DNA. If necessary can digest parent strands, but after enough cycles the majority should be mutated anyways.
what would you observe in the clones of the genomic library versus the cDNA library?
the genomic library will not give gene products because eukaryotic DNA has introns and the prokaryotic system will not slice the RNA. used to observe the DNA. the cDNA will give gene products, because cDNA only contains the sequence contained in mRNA. used to observe gene products.
Discuss the selection of primers and the three repeated steps in the process: Heating, Cooling, and Replication. Describe what happens in each of these three steps.
the primers should be oligonucleotides (20-25 nucleotides) that anneal to 3' end of desired sequence of DNA (then read 3'-5' from that point, synthesize 5'-3') steps: -heating: denatures DNA -cooling: anneal primers to DNA -replication: introduce DNA poly, replicate DNA
Transformation
the process of introducing small plasmids into bacterial cells achieved by putting the cell under stress (heat shock, high/low pH, electrophoresis), if incubated with plasmids cells uptake plasmids
Micro-RNAs can be used to....
visualize a "knock out" phenotype without inactivating a gene
describe how the yeast two-hybrid analysis is used to identify interacting proteins.
yeast has Gal4p protein that activates transcription of Gal genes. Has 2 domains that are stable when separated, but are required together for transcription activation. experimentally, fuse one domain to protein A, other domain to protein B. When together, if proteins A and B interact, they will bring the domains together and transcription will be activated. If they do not interact, no transcription (can mate protein A strain with an entire library and identify which proteins it interacts with)
