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three steps in initiation of replication by DnaA
(A) DnaA protein binds first to the DnaA boxes (both high and low affinity sites) forming a filamentous structure. (B) As more DnaA binds, the filament extends into the DNA unwinding element, which then becomes unwound. (C) Two complexes of DnaB and DnaC bind to the DNA unwinding element. This eventually pushes DnaA away and opens up the whole AT-rich region. The two DnaB complexes form the basis for two replication forks, headed in opposite directions around the circular DNA
bridge amplification
- When a DNA fragment on an Illumina colony with attached ends binds to other identical attached ends in the same colony. The sequence between these attached ends is duplicated (forming a bridge between the two) and then the ends are released and they bind to other similar ends.
Bridge Amplification
- When a DNA fragment on an Illumina colony with attached ends binds to other identical attached ends in the same colony. The sequence between these attached ends is duplicated (forming a bridge between the two) and then the ends are released and they bind to other similar ends. The individual genomic DNA strand attached to the flow cell does not provide ample enough signal for sequencing to occur, therefore, it must be converted into a group or cluster of identical copies via PCR. The forward and reverse primers for PCR are attached to the flow cell surface, and therefore, the genomic DNA arches over during annealing so that DNA polymerase can make a copy of the strand.
Process of PCR
- denaturation (94): denature template DNA by heat, strands separate cause h bonds break - annealing (variable): cool to allow primers to form h bonds at beginning of gene with ends of target sequence of gap - extension (72): DNA polymerase adds nucleotides to 3' end of each primer run a gel after ~30 cycles
How does telomerase work?
-Telomerase binds to the telomere through base pairing between RNA subunit and overhang of parent strand (this extends the parent strand which allows an RNA primer to be created which can then be extended to fill in the lagging strand)
how much dna can one make in a single pcr reaction?
1 copy, 500 bp = 5x10^-10 ng of DNA
how his pcr used to create linear fragments of dna that have ends homologous to regions of bacterial artificial chromosomes? (recombineering)
1. Bacteria containing the genes for the Red proteins are grown at a high temperature for a few hours to induce production of the Red proteins, 2. which then recognize the ends of the insert and the region on the vector that has the homologous sequence. 3. The enzymes cut the vector DNA, and attached the insert. 4. The final bacteria are grown at 32°C to inhibit any further Red gene expression.
shotgun sequencing steps
1. Break multiple copies of genome into smaller, overlapping random fragments 2. Sequence each fragment and assemble genome based on overlapping sequences - Issue: Reassembly process can be derailed by repetitive nucleotide sequences (rare in bacteria, but a large fraction of vertebrate genomes) -- when sequence assembled incorrectly, intervening info lost --- To avoid: combined with clone-by-clone approach
steps of intiattion
1. bacterial cell with circular chromosome 2. newly replicated DNA 3. daughter chromosomes attached to membrane 4. septum forms chromsomes in daughter cells
how does TOPO TA cloning work?
1. the terminal transferase activity of Taq adds an extra adenine at the 3′ end of the PCR product. 2. The TOPO-TA cloning vector has single 5′ thymidine overhangs at each end, which is covalently attached to the topoisomerase I enzyme. 3. When the 5′ end of the insert nears the 3′ T overhang, the energy from the covalent bond transfers to connect the insert and vector.
process of combining an insert and vector
1. vector and insert are cut with same RE to create compatible single strand DNA overhangs 2. mixed together with DNA ligase which jpins the ends yielding a closed double stranded plasmid carrying the gene of interest
sequences at ORI
13bp repeats high affinity DnaA box low afifnity DnaA box
TOPO TA cloning
3'-A tailed double-stranded DNA fragment; TOPO vectors with topoisomerase I covalently bound to vector(T-P-topoisomerase at 3' end) the 5'-OH group of the DNA fragment can attack the TOPO vector and release the topoisomerase I enzyme, produce a ligated circular vector
how much can you detect of pcr with a gel?
<1 ng
replication fork
A Y-shaped region on a replicating DNA molecule where new strands are growing. The assembled proteins, known as the replisome, facilitate the unwinding of the helix and the addition of new nucleotides. The arrows indicate the direction of movement of the replication fork. The synthesis of two DNA helices results from adding a new complementary strand to each one of the separated old strands
sliding clamp loader
A component of the DNA polymerase III holoenzyme consisting of five subunits that attach a circular protein complex to the polymerase in an ATP-dependent reaction.
selectable marker
A gene, such as one encoding resistance to an antibiotic, that can be used to identify (select) cells that contain recombinant DNA from among a large population of untransformed cells.
Taq polymerase
A heat-stable form of DNA polymerase extracted from bacteria that live in hot environments, such as hot springs, that is used during PCR technique
immunological screening of DNA library
Bacteria carrying a library are grown on agar, transferred to a membrane, and lysed. Released proteins are bound to the membrane. These proteins include both those from the library as well as many bacterial proteins. The membrane is incubated with a primary antibody that only binds the protein of interest. Any nonspecifically bound antibody is washed away. Finally, a second antibody that binds the primary antibody and that also carries a detection system is added.
what bacteria are associated with metabolic disease?
Bacteroidetes
electroporation process
Before the electrical pulse bacteria and completed vector with insert are separate. During the electric pulse small pores open in the bacterial cell membrane, and the vectors with insert can enter into the cytoplasm. After the pulse, the bacterial cell membrane recloses, capturing the vector inside the cell. Electroporation can also move vectors into mammalian cells and yeast cells
dna library
Collection of cells that host different fragments of foreign DNA, often representing an organism's entire genome.
shotgun sequencing
Cutting DNA into random fragments and then determining the sequence of bases in each fragment
Metagenomics
DNA from a group of species is collected from an environmental sample and sequenced
what does it mean that dna is semi conservative
DNA is replicated semiconservatively; that is, each parental DNA is separated into two strands and copied. The two daughter molecules have one old strand from the parent and one newly synthesized strand.
dna synthesis - phosphodiester bonding
DNA polymerase links nucleotides via phosphodiester bonds. DNA polymerase breaks the bond between the first and second phosphates of the incoming deoxynucleoside triphosphate, which is then joined to the free 3′ hydroxyl of the growing DNA chain. The two outer phosphates of the dNTP (called pyrophosphate) are released from the reaction, as well as, a hydrogen ion (H+) from the 3′ -OH group.
sequencing by synthesis (Ion torrent)
DNA polymerase, PCR primers, and one of the four dNTPs are washed over the surface of the semi-conductor chip. If the genomic DNA fragment has the complementary base next to the 3′ end of the sequencing primer, DNA polymerase will incorporate the single dNTP. In the top left, dGTP was incorporated. As the phosphodiester bond forms, a proton (H+) is released, which changes the pH of the nanowell (top right). The pH change is converted to a change in voltage, which is recorded as 1. In contrast, when the genomic DNA fragment does not have the complementary base to the added dGTP (bottom row), then the pH does not change. The change is voltage is recorded as a 0.
degenerate dna primer design
Degenerate primers can be designed based on a short amino acid sequence from a protein. Because many amino acids are encoded by several alternative codons, the deduced DNA coding sequence is ambiguous. For example, the amino acid tyrosine is encoded by TAC or TAT. Hence, the third base is ambiguous, and when the primer is synthesized a 50:50 mixture of C and T will be inserted at this position. This ambiguity occurs for all the bases shown in red, resulting in a pool of primers with different, but related sequences. Hopefully, one of these primers will have enough complementary bases to anneal to the target DNA sequence that corresponds to the gene for the protein.
Sanger sequencing method
Divided into 4 samples (ddA, T, G, C) Label with radioactive/dye oligo at 3' end Mix with taq, dNTPs, ddNTP and incubate run on gel --> frags will terminate at different lengths
dna synthesis - priming anf elongation process
During normal DNA synthesis, DNA polymerase reads the template DNA and makes a new complementary strand of DNA. To get DNA synthesis started, a short oligonucleotide primer must anneal to the beginning of the target DNA. DNA polymerase recognizes the 3′ end of the primer and connects the 5' end of the incoming nucleotide; hence, synthesis occurs in a 5′ to 3′ direction.
what do you stain PCR with in a gel?
EtBr
(T/F) RT-PCR stands for real-time PCR
F, reverse transcriptase
transfection method: viruses
First the mammalian vector must be packaged into the virus head (reddish circle with spikes). The virus with the mammalian vector attach to the cell and enter via endocytosis.
cDNA library process
First, eukaryotic cells are lysed and the mRNA is purified. Next, reverse transcriptase plus primers containing oligo(dT) stretches are added. The oligo(dT) hybridizes to the adenine in the mRNA polyA tail and acts as a primer for reverse transcriptase, which synthesizes a DNA strand complementary to the mRNA. DNA polymerase is added to synthesize the opposite DNA strand, thus creating double-stranded cDNA. S1 nuclease trims off singlestranded ends. Since each mRNA has a different sequence, linkers must be ligated to the ends of the cDNA to allow convenient insertion into the cloning vector. After addition, the linkers are digested with the appropriate restriction enzyme and the cDNA is ligated into the vector. The resulting hybrid DNA molecules are then transformed into bacteria, so giving the final cDNA library.
Synthesis of precursors
First, ribonucleotide reductase converts the ribonucleoside diphosphates to the deoxy form. Second, a kinase adds a phosphate to form the deoxyribonucleoside triphosphates. The precursor for the thymidinenucleotides is made from a uridine derivative by adding a methyl group that is transferred by the carrier tetrahydrofolate (THF).
Sequencing by Synthesis (Illumina)
For each cluster of DNA on the flow cell, a supply of sequencing primers, fluorescently-labeled dNTPs, and DNA polymerase are added (left). As the temperature is adjusted the primer anneals to the DNA in the cluster, and DNA polymerase attaches. Only one DNA strand from this cluster is shown for clarity, but in reality the entire cluster of sequences are decoded simultaneously. After the first complementary nucleotide is added to the end of the primer, the fluorophore blocks DNA polymerase from adding any more nucleotides. The computer records the identity of the fluorophore, which is then removed and washed away. DNA polymerase adds the second complementary nucleotide, the identity is recorded and the fluorophore is removed. These cycles repeat to get a read from this cluster.
how does replication fork proceed?
For the replication fork to proceed, both the double helix and the supercoils must be unwound. Helicase unwinds the double helix and DNA gyrase removes the supercoiling.
what separates s and m phase
G1 and G2
Gibson Assembly process (c) fragments
Gibson assembly can assemble multiple fragments, including the vector sequence. The example shows the three insert fragments joining with the vector.
gib assembly yellow
Gibson or isothermal DNA assembly combines DNA fragments with overlapping sequences by first making each end single-stranded with a DNA exonuclease. The overlapping complementary ends anneal spontaneously, any single-stranded gaps are filled with DNA polymerase, and the backbones are sealed with DNA ligase.
template strand
Incoming nucleotides line up on the antisense or template strand and are then linked together to form the new strand of DNA. The arriving nucleotides are positioned by base pairing in which A pairs with T and G binds to C. These base pairs are held together with hydrogen bonds.
Eukaryotic Cell Cycle
Interphase (g1, s phase, g2 phase) and then Mitotic phase (mitosis, cytokinesis)
what bacteria are primarily in vagina?
Lactobacillus
semi-conservative replication
Method of DNA replication in which parental strands separate, act as templates, and produce molecules of DNA with one parental DNA strand and one new DNA strand
ion torrent NGS
Microbeads coated with oligonucleotides (pink and yellow) complementary to the adapters are combined with genomic DNA library fragments so that one fragment binds to each bead.
replication bubbles
Multiple sites of replication found on large, linear eukaryotic linear eukaryotie chromosomes. Numerous openings in the DNA, or replication bubbles, occur at the sites of replication in eukaryotic chromosomes. The longer replication continues, the larger the bubbles. The bubbles eventually merge together, which separates the newly replicated DNA molecules (not shown).
PCR: Extension/Elongation
Once the primers have annealed to the template, the temperature is increased to the optimal temperature for thermostable DNA polymerase, typically around 72°C. The polymerase synthesizes a complementary DNA strand to the template DNA using the pool of dNTPs as building blocks for the new DNA and using the 3′ end of the primer as a starting point. Extension will continue past the end of the target sequence for the first few cycles of PCR. Although the PCR primer is not synthesized, it is incorporated into the final new DNA strand without any modifications
how does rna primer work?
One RNA primer is needed to start the 5' to 3' leading strand. In contrast, multiple RNA primers are needed for the 3' to 5' lagging strand because this is made in short stretches each running 5' to 3'. DNA polymerase will then add nucleotides to the end of each RNA primer. Later, the short RNA primers will be removed and replaced by DNA.
how are precursors for dna made?
Precursors for DNA are made from ribonucleotides by oxidizing the ribose to deoxyribose.
tailed pcr primer
Primers for PCR can be designed to have non-complementary regions at the 5′ end. After PCR, the final PCR amplicon will have the 5′ ends added onto the target DNA sequence.
3 steps in starting a primer for a new okazaki fragment
Prior to primer formation, the bases of the parental DNA strand are covered with SSB proteins. (A) First, the PriA protein displaces the SSB proteins. (B) Second, primase associates with the PriA protein. (C) Lastly, the primase makes the short RNA primer needed to initiate the Okazaki fragment
how do quinolone antibiotics work?
Quinolone antibiotics kill bacteria by inhibiting DNA gyrase. This blocks replication of the DNA.
sequences at the origin of replication (ORI)
Sequence repeats at the origin of replication are of two varieties, both being AT-rich. The three 13-base repeats together comprise the DNA unwinding element. Nearby is located a cluster of DnaA binding sites, three of high affinity and several more of lower affinity.
advantage of shuttle vector
Shuttle vectors can replicate in more than one organism. This allows the same gene to be expressed in different hosts
T/F RT-PCR can determine the amount of mRNA for a particular gene in two different growth condition
T
T/F expression vectors can have tightly regulated promoters
T
T/F in pcr how long each step lasts is also variable, but the total time for all three steps is about 1-2 minutes. Thus, a PCR reaction, start to finish, is 30-60 minutes.
T
t/f Shuttle vectors must have separate origins of replication and separate selection mechanisms for each compatible host organism.
T
where terminator sequence why?
Terminator sequences are positioned at the end of the MCS in order to stop transcription of the gene of interest.
Gibson Assembly process (a) fragments
The DNA fragments for a large insert are shown. These three fragments were created by PCR using tailed primers. The tail region of the primers created the overlap region that is identical to the adjacent fragment.
active replication complex
The MCM helicase assemblies in the pre-replicative complex are activated by phosphorylation. This allows binding of Cdc45 and several Sld proteins. Sld2 and Sld3 are activated by phosphorylation, which enables binding of the GINS complex. (Binding of Mcm10 and recruitment of DNA polymerase then triggers separation of the assembly into two replisomes that move in opposite directions—not shown).
Mammalian expression vector components
The MCS or multicloning site is flanked by a promoter (CMV promoter) that is from a virus, but functions in the mammalian cell and a polyA tail sequence. The NeoR gene is also flanked by a promoter that functions in mammalian cells and a polyA tail signal sequence.
how is YAC used in bacteria?
The circular form can be manipulated and grown like any other plasmid in bacteria since it has a bacterial origin of replication and an antibiotic resistance gene.
DNA poly III proofreading
The component of DNA polymerase responsible for checking the accuracy of new nucleotides is the DnaQ protein (ɛ-subunits). A mismatch is detected by a bulge in the new chain. The incorrect nucleotide is discarded and the appropriate nucleotide added.
metagenomicss human biome
The composition of the human microbiome varies greatly between different body locations. The pie diagrams illustrate the overall make up of these bacterial populations. Note that the gut microbiome outnumbers all the others combined. A variety of diseases that are known to correlate with microbiome composition are indicated. In most cases, it is not known to what extent the presence of particular bacteria causes the condition or are the result of it
fluorophore of bases
The dideoxynucleoside triphosphates for G, A, T, and C have a different fluorophore attached to their structure. Typical sequencing reactions show the color of these fluorophores as black for G, blue for C, red for T, and green for A, even though, these are not the actual colors for the fluorophores.
eukaryotic replisome
The eukaryotic replication fork is created by MCM helicase unwinding the two strands of DNA, and RPA coating these to prevent reannealing. Next, the DNA polymerase α complex makes an RNA primer followed by iDNA. Next, the RFC clamp-loader complex assembles the sliding clamp (PCNA) around the single-stranded DNA and recruits another DNA polymerase. DNA polymerase ɛ loads onto the leading strand, and DNA polymerase δ loads onto the lagging strand to synthesize new DNA from the 3′ end of iDNA. On the lagging strand, sections of RNA primer followed by iDNA are removed by an exonucleaseand then replaced with new DNA by DNA polymerase δ
first step of joining okazaki
The first step in replacing the RNA primer with DNA is the binding of DNA polymerase I to the primer region. As Pol I moves forward it degrades the RNA and replaces it with DNA. Lastly, DNA ligase seals the nick that remains
Gibson Assembly process (b) fragments
The fragments are then mixed with three enzymes, a DNA exonuclease that degrades the 5′ ends of each fragment, leaving 3′ single-stranded DNA overhangs. Since the sequences are identical at the ends, these overhangs are complementary and anneal. Next, any regions of the DNA that are still single-stranded are filled in by the second enzyme in the mixture, DNA polymerase. Finally, the third enzyme, DNA ligase, connects the backbones of all the fragments to create one continuous seamless piece of DNA. Notice that if the vector was included in the assembly, it would also anneal to the insert.
dna library process
The genomic DNA from the organism of interest is isolated and digested with a restriction enzyme. Usually, the restriction enzyme has a recognition sequence of four bases, and the DNA would be cut into fragments much smaller than the average gene. Therefore, the digestion is carried out for a brief period that leaves many of the restriction sites uncut, called a partial digest. A suitable vector for the required insert size is chosen and is cut with a restriction enzyme that produces compatible sticky ends. The digested genomic DNA and the vector are ligated together and transformed into bacterial host cells. A large number of transformed bacterial colonies must be isolated and kept to ensure that all possible genes from the genome of interest are represented on at least one vector
Read depth in NGS
The greater number of reads, the more confidence in the identity of that particular base. In the position that has a read depth of two, the base could either be a G or C.
insertional inactivation
The inserted DNA inactivates a gene in the vector by inserting into that gene. This allows cells which contain the recombinant DNA molecule to be identified.
pre-replicative complex
The origin of replication successively binds the origin recognition complex (ORC) and the Cdc6 protein. Together with Cdt1, these then recruit two MCM helicase complexes. This forms the pre-replicative complex (pre-RC).
capillary electrophoresis
The smaller fragments travel faster through the tube, and the larger fragments travel slower. As the fragments pass through the laser, the fluorophore at the 3′ end of the fragment emits the characteristic color of light for the base. In this example, the T is labeled with a fluorophore that emits blue light. The detector then records the color, and reports the color as a T. Therefore, the first decoded nucleotide for the target DNA is a T. As the next fragment moves into the laser light, the final nucleotide will emit yellow light, and this is recorded as an A. The final results are compiled as sequence files for analysis
PCR second cycle
The two DNA pieces produced in the first cycle are denatured, primers annealed to their complementary sequence in the target DNA, and then DNA polymerase extends the primers to create four double-stranded DNAs. Newly made DNA is shown in blue, and single-stranded DNA from previous cycle is shown in pink.
How is PCR used in forensics?
The two PCR primers will amplify the target DNA from a crime scene or reference sample if the DNA has complementary sequences to the primer. In this example, the first unknown sample did not have complementary DNA sequences, and the PCR primers did not produce any PCR amplicons. In the second unknown sample, the DNA had complementary DNA samples, and therefore, the primers were able to anneal so that DNA polymerase could make copies.
beta galactosidase structure (alpha complementation)
The two protein fragments can be encoded on two different molecules of DNA within the bacterial cell. The alpha fragment can be expressed from a plasmid and the remainder of the β-galactosidase can be expressed from the chromosome.
how is mammalian expression vector maintained?
The vector is not maintained in mammalian cells though. Instead, the vector is propagated in bacterial cells, and therefore contains a bacterial origin of replication and a selectable marker (AmpR) that is for growth in E. coli.
Sanger sequencing method
There are 4 test tubes each one containing ddNTP (only one), dNTP (all 4), DNA Polymerase, Primer, ssDNA template, Mg. Synthesis occurs untill a ddNTP is incorporated. You use gel electrophoresis to see the fragements. Read the gel bottom to top for the sequence.
why is it hard to align reads from repeated regions?
This read could be aligned to either location in the genome.
expression vector for lac promoter. how work?
To stimulate transcription, an artificial inducer molecule called IPTG is added. IPTG binds to the LacI repressor protein, which then detaches from the DNA. This allows RNA polymerase to bind. Before IPTG is added to the culture, the LacI repressor prevents the cloned gene from being expressed.
how does helicase unwind?
To unwind DNA, helicase first binds to DNA and then cleaves the hydrogen bonds connecting base pairs to separate the strands of the helix. Then single-stranded binding protein attaches to the single-stranded DNA.
ultra-sonic waves -DS DNA
Ultrasonic sound waves (gray wavy lines) focused on the DNA sample (pink), create small gas bubbles in the liquid surrounding the DNA. As the bubbles collapse, the force breaks apart the covalent bonds holding the DNA backbone together. This releases smaller DNA fragments. The sheared DNA is randomly broken so every fragment ends in different locations of the genome.
TA cloning
Use DNA polymerases to add a single 3'-A residue to double-stranded DNA fragment via a terminal transferase-like activity; the A-tailed DNA product is ligated with a linearzed vector with 3'-T overhangs by DNA ligase. (PCR product with a single template-independent base addition of an adenine (A) residue to the 3' end of the PCR product, through the normal action of the polymerase. These "A-tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase, followed by transformation)
hairpin structure pcr
When sequences within a single PCR primer are complementary, then the primer will fold back on itself to form a hairpin.
Real-time PCR (qPCR) with sybr green
When the fluorescent dye SYBR Green I is present during a PCR reaction, it binds to the double-stranded PCR product and emits light at 520 nm. The SYBR Green dye only fluoresces when bound to DNA, therefore, the amount of fluorescence correlates with the amount of PCR product produced. The accumulation of PCR product is followed through many cycles by measuring the amount of fluorescence in each cycle, and plotted on a graph.
yeast artifical chromosome (YAC) has two forms. what are they?
a circular for bacteria, linear for yeast
A shuttle vector
a cloning vector that can replicate in two or more dissimilar hosts
Replisome
a complex of DNA polymerase and other enzymes that catalyzes the synthesis of DNA
If essential genes are removed from a virus vector what is needed
a helper virus is needed for viral replication.
Describe how restriction enzymes make it possible to make recombinant plasmids
a restriction enzyme cuts at specific sequences. a RE cuts both the vector and insert to create compatible ssDNA overhangs. the end are then put back together using dna ligase. thus allowing for a new recombinant plasmid to be formed
first two steps of pcr (denaturing the template and primer annealing)
a small amount of template DNA is heated to 95°C, which separates the two strands of the double helix. When the temperature is lowered to approximately 60°C, the primers anneal to the ends of the target sequence. Since the primer is present in large excess over the template DNA, the majority of template strands will bind to primers rather than re-annealing to each other. Notice that the PCR primers anneal so they are antiparallel with the target DNA.
how to screen for inserts with a LacZalpha gene? blue/white screening of plasmid inserts
a small polylinker or multiple cloning site is inserted into the extreme N-terminal portion of lacZα. The small insertion does not affect the function of the alpha fragment, and is still active when complexed with the remainder of β-galactosidase expressed from the chromosomal gene. Bacteria containing this construct will turn blue in the presence of X-gal. However, if a large segment of DNA, such as a cloned gene, is inserted into the multiple cloning site, the alpha fragment is disrupted and β-galactosidase is no longer active. Bacteria harboring this plasmid cannot cleave X-gal, and therefore remain white.
DNA polymerase III
adding bases to the new DNA chain; proofreading the chain for mistakes the sliding clamp loader makes contacts with single-stranded binding proteins and the sliding clamps in order to stabilize the template DNA as it enters into DNA polymerase III, which polymerizes new DNA as the template passes.
what diseases are associated with skin microbiome?
allergies acne psoriasis atopic dermatitis ectodermal dermatitis skin cancer
what can affect your gut microbiome?
antibiotics host factors diet environment
why cant DNA polymerase add another nucleotide to a chain ending in dideoxyribose
because its 3′ carbon does not have a hydroxyl group.
how are mrnas features used to isolate itself?
by purifying with OligodT Only mRNA has a polyA tail, a long stretch of adenines following the coding sequence. The polyA tail of the mRNA will bind by base pairing to an oligonucleotide consisting of a long stretch of deoxythymidine residues—oligo(dT). The oligo(dT) is attached to glass or magnetic beads, which consequently bind mRNA specifically. Other RNAs will not bind to the beads and can be washed from the column.
transfection methods
calcium phosphate precipitation lipid droplets viruses
why is beta galctosidase unique?
can be expressed as two pieces that come together to form functional protein
sanger: how to seperate fragments?
capillary electrophoresis
ColE1
carries a gene for colicin E1 - a protein that kills bacteria not carrying this plasmid
expression vector
contains sequences upstream of the cloned gene that control transcription and translation of the cloned gene
multiple cloning site
contains the recognition sequence of several different restriction enzymes
multiplex pcr
creates targeted sequencing library Simultaneously amplifying many different regions of a genome using different PCR primer sets creates a series of PCR amplicons for just the genes or regions of interest in the genome.
Sanger dideoxy sequencing
ddNTP are used in place of dNTP (they are marker nucleotides). When ddNTP molecule taken up, replication of new DNA molecule ceases. We can look at where the DNA fragment stops & correspond that to a specific nucleotide. This allows us to sequence the DNA.
Directed mutagenesis
deliberate alteration of the DNA sequence of a gene by any of a variety of artificial techniques
Discuss the different DNA polymerases in the bacterial replisome - Names of each What they do
dna poly I - removes rna primers from lagging strand dna poly III - major polymerase used in replication dna poly V. replicates damaged DNA in the SOS repair system
Discuss the different DNA polymerases in the eukaryotic replisome - Names of each - What they do
dna polymerase Alpha - responsible for initiation of new strands. First, the complex makes an RNA primer. Then, polymerase α elongates the RNA primer by making a short piece of DNA about 20 nucleotides long (iDNA) dna polymerase epsilon - loads on the leading strand. used to make the leading strand dna polymerase delta - loads onto lagging strand to synthesize new dna from the 3' end of iDNA dna polymerase beta - removes and repairs altered and defective nucleotides
Promoters are highly variable because
each host organism has different structural requirements in order to recognize the promoter, and because the promoter allows the researcher to control the expression of the gene of interest.
ion torrent NGS: what seperates each bead into water droplet?
emulsion When the beads are mixed with water and oil, each bead is encapsulated in a separate water droplet that prevents the PCR reactions from cross contaminating other beads.
Strong promoters are used to what?
express high levels of proteins from cloned genes.
T/f RT-PCR doesnt require primer
f
transfection methods: lipid droplets
get into mammalian cells by attaching to lipid droplets (purple/pink circles) that have positive charges on the outer surface. The positively charged droplets attract the negatively charged DNA and the lipid portion then fuses with the cell membrane to release the DNA into the cell.
The origin of replication controls
how many copies of the plasmid are present in each bacterial cell and the mode in which the plasmid is replicated. Bacteria can maintain more than one type of plasmid if the two have different origins of replication.
Emulsion PCR
isolates individual DNA molecules with primer coated beads in an oil phase, PCR coats beads in clonal copies of DNA molecules
what does beta galactosidase cleave?
lactose into 2 monosaccharides ONPG and x-gaI -> releases a visible dye. ONPG = yellow, X-gai blue
Recombineering uses what to connect the ends of the insert with the homologous DNA sequence on the vector.
lambda phage enzymes, called Red,
the 5' end is potentially ____ in replication of lienar dna. WHy?
lost. When an RNA primer is removed after initiating a strand of linear DNA the gap cannot be filled by DNA as there is no upstream 3′-hydroxyl to accept nucleotides. Thus, linear DNA would be shortened during each replication cycle.
Thermocycler
machine that raises and lowers temp of DNA in precise intervals for PCR
what bacteria are in placenta microbiome?
mainly proteobacteria
what diseasea are associated with the gut microbiome?
metabolic syndrome diabetes c.difficile infection colorectal cancer iBD psychiatric disorders
transfection methods: calcium phosphate
mixes the mammalian vector DNA (blue circles) with calcium phosphate to create a precipitate. After the mammalian cells are coated with the precipitate, the DNA is taken into the cell via endocytosis or phagocytosis.
Gibson assembly can connect what
multiple DNA fragments into one without adding any extra base pairs of DNA between the insert and vector.
When first made, the lagging strand is composed what
of alternating Okazaki fragments and RNA primers.
RT-PCR primers
oligodT - Entire mRNA samples can be converted into cDNA using oligo(dT) primers that anneal to the polyA tail of mRNAs. The cDNAs will tend to have sequence that is from the 3′ end of mRNAs. random hexamers - (B) Since random hexamers primers have every potential six-base sequence, the final cDNA sample will have complementary sequences from random parts of every mRNA in the sample.
polymerization of nucleotides
phosphodiester bonds join sugar-phosphate backbone At each step, a nucleoside triphosphate is added. During chain elongation, the two outermost phosphate groups of the precursor are cleaved off, releasing pyrophosphate. This provides energy for the reaction. The 3′-OH group of the precursor triphosphate remains available for the next addition to the growing strand.
A ________ ______ has many available restriction sites to ease the process of cloning foreign DNA fragments.
polylinker region
what diseasea are associated with the placental microbiome?
preterm birth chorioamnionitis vilitis torch infections
what can go wrong when designing primers?
primer dimers hairpin structures
Vectors may carry what to mediate the expression of cloned genes.
promoters and ribosome binding sites
what bacteria is primarily in skin?
proprionibacterium
why was amp resistance added to ColE1?
provides an easily identifiable bacteria that carry the altered plasmid
ONPG
releases o-nitrophenol yellow
x-gaI
releases unstable group that reacts with oxygen to form blue color
Illumina sequencing
removable terminators are used to sequence many DNA fragments simultaneously
what was done to original ColE1 plasmid?
remove colicin gene, add amp resistance
The fragments of the discontinuous lagging strand must be finished by what
removing the RNA primers (RNase H or Pol I), filling the gaps with DNA (Pol I) and, finally, joining the ends (DNA ligase).
fecal transplant
restores gut health in individuals with Pseudomembranous colitis (caused by clostridium difficile)
primer dimers
result from binding of primers onto each other through short (2-3 base) homologies at 3' ends & copying of each primer sequence If the sequence between two PCR primers is complementary, then the primers preferentially bind to each other rather than the target DNA to form a primer-dimer.
what if need to amplify rna?
reverse transcriptase pcr
strand initiation requires what?
rna primer
when does dna replication occur cell cycle? when do chromosomes separate?
s phase m phase
whats in the test tube, pcr? how much?
sample buffer dNTPs primers DNA polymerase 20-50 uL
Sanger method
selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
The term "vector" refers to
selfreplicating DNA molecules used to carry cloned genes (or any other pieces of cloned DNA).
rna primer
short segment of RNA used to initiate synthesis of a new strand of DNA during replication
Describe the features of a plasmid. At least 5
small, circular replicate independently of host's chromosomal DNA provide functional benefits such as antibiotic resistance usually have selectable marker, promoter, 5' primer site, restriction sites, 3' primer site, and origin of replication size range of 1-200 kb
why are polylinkers designed that way?
so that the restriction enzyme sites is unique, and the corresponding enzymes only cut the plasmid once.
what dn polymerase can survive near boiling temperatures (94C)
taq polymerase
what replaces repeats at the ends of chromosomes
telomerase Telomerase RNA recognizes the tandem repeats at the end of linear DNA. The RNA of telomerase sticks out beyond the chromosome ends and serves as a template for addition of new DNA repeats that will repair the segment lost during the last DNA replication.
PCR: products from 3rd cycle
the PCR amplicons or PCR products from the second cycle go through the same process as before. The four double-stranded pieces are denatured into eight singlestranded pieces (pink strands). The primers anneal and DNA polymerase makes the complementary strands. This cycle has the first double-stranded copy of the target region without any single-stranded overhangs. Newly made DNA is shown in blue
what determines annealing temperature? what happens if temp too low?
the PCR amplicons or PCR products from the second cycle go through the same process as before. The four double-stranded pieces are denatured into eight singlestranded pieces (pink strands). The primers anneal and DNA polymerase makes the complementary strands. This cycle has the first double-stranded copy of the target region without any single-stranded overhangs. Newly made DNA is shown in blue
elongaiton of the cell seperates what? causes what to form>
the chromosomes Segregation of chromosomes is caused by elongation of the cell. Subsequently, a partition, or septum, is formed that completes cell division.
how is YAC used in yeast?
the circular form is isolated and linearized such that the yeast telomere sequences are on each end. This form can accommodate up to 2000 kb of cloned DNA inserted into its multiple cloning site (MCS)
what happens after ultra-sonic waves?
the ends are uneven. T4 DNA polymerase fills in any single-stranded regions with complementary nucleotides. Then Klenow polymerase adds a single A onto the 3′ end with its terminal transferase activity. The single A overhang facilitates the addition of the adapter, which is synthesized to have a single T overhang. Finally, DNA ligase connects the adapter to each end of the genomic DNA fragment.
When Taq polymerase amplifies a piece of DNA during PCR what happens?
the terminal transferase activity of Taq adds an extra adenine at the 3′ end of the PCR product. The TA cloning vector was designed so that when linearized it has single 5′ thymidine overhangs at each end. The PCR product can be ligated into this vector without the need for special restriction enzyme sites
Where is taq polymerase from?
thermus aquaticus
why is annealing temperature variable?
to avoid mispriming
how are mammalian cells taking up expression vectors?
transfection
adapter features
two different adapters are added onto the ends of genomic DNA fragments during NGS library preparation. The left adapter and right adapter have a sequence complementary to another oligonucleotide that is attached to the solid support, an index or barcode sequence, and binding site complementary to the sequencing primers. These sequences vary between left and right adapters and will vary depending upon the type of NGS sequencing methodology used.
what must a shuttle vector have for yeast and ecoli
two origins of replication, one for E. coli and one for yeast; a yeast centromere sequence so that it is partitioned into the daughter cells during yeast replication; selectable markers for both yeast and E. coli; multiple cloning site for inserting the gene of interest.
process of pcr - detailed
two primers anneal to complementary sequences at either end of a target sequence on a piece of denatured sample DNA. DNA polymerase synthesizes a complementary strand of DNA from the primers, resulting in two new strands of DNA. In further cycles, the newly made DNA molecules are denatured, primers are annealed, and DNA polymerase copies the target regions, resulting in multiple copies of the original target sequence.
Discuss RT-PCR (reverse transcriptase) Why and how
two-step procedure that involves making a cDNA copy of the mRNA, then using PCR to amplify the cDNA. First, a sample of mRNA is isolated. Reverse transcriptase is used to make a cDNA copy of the mRNA, which is then amplified by a thermostable DNA polymerase in a regular PCR reaction.
reverse transcriptase pcr (RT-PCR)
two-step procedure that involves making a cDNA copy of the mRNA, then using PCR to amplify the cDNA. First, a sample of mRNA is isolated. Reverse transcriptase is used to make a cDNA copy of the mRNA, which is then amplified by a thermostable DNA polymerase in a regular PCR reaction.
what diseases are associated with the vaginal microbiome?
vaginosis stds yeast infection
After 30 cycles of PCR, how many copies of the target sequence can potentially be produced from a single starting molecule of DNA?
~1.1x10^9 copies
how many cycles are usually in pcr?
~30
