Module 3 Sections 1-5 Flashcards

Question

ionic interactions

Answer 1

Negative charge of backbone phosphates are neutralized by interactions with cations Na+ and Mg++ commonly interact with the backbone to neutralize the electrostatic repulsion between strands

Answer 2

1. temperature above melting temp makes DNA unwind to single strand, destablizing it 2. salt increase in salt conc = increase in duplex stability sodium ions interact with the negatively charged DNA backbone and stabilizes it 3. proteins DNA binding proteins are involved in the compaction of genomes and contribute to both the global and local structure of DNA 4. organic solvents Carbon-based Destabilize DNA helix by disrupting hydrogen bonds and solvating bases

Answer 3

Due to the negative charge of the sugar-phosphate backbones of both nucleic acids Backbone of both DNA and RNA is hydrophilic, so the hydroxyl groups of the sugar residues form hydrogen bonds with water

Answer 4

have pKA near 2 completely ionized negatively charged (neutralized by ionic interactions with positive charges on proteins, metal ions, or short linear organic molecules called polyamines)

Answer 5

2 or more amine groups

Answer 6

Transient carriers of genetic information Transcript copy of a gene that encodes a specific protein Carries the encoded information from the nucleus to the ribosomes where the protein is produced Coding sequence of mRNA determines the amino acid sequence of the protein Different mRNA molecules adopt different 3D structures depending on what is most energetically favourable

Answer 7

Transfer RNA (tRNA) - Present during translation Ribosomal RNA (rRNA) - Present during translation Long non-coding RNAs (lncRNA) - Can be important regulatory RNAs Small nuclear RNAs (snRNAs) - Play a role in gene regulations (splicing "snurps") MicroRNAs (miRNAs) - Limit translation by binding to the 3'-end of target mRNAs Small interfering RNAs (siRNAs) - Can inhibit transcription of certain genes and viral DNA Catalytic RNAs - Ribozymes

Answer 8

both carriers of genetic information

Answer 9

DNA - long term storage of genetic information RNA - more transient (less permanent) DNA - set of biological blueprints RNA - helps carry out the guidelines found within these blueprints DNA - double stranded RNA - single stranded DNA - not as versatile RNA - structurally and functionally versatile

Answer 10

called deoxyribose In RNA, the deoxyribose sugar is replaced with ribose Ribose is a 5-carbon sugar with a hydroxyl group at the 2' carbon. This provides an additional site for hydrogen bonding, stabilizing the 3-dimensional folding of the polynucleotide

Answer 11

RNA has uracil instead of thymine

Answer 12

RNA can sometimes have A-A and G-U base pairing (helps stabilize the 3 dimensional folding of RNA, with surfaces capable of binding other molecules)

Answer 13

under alkaline conditions (pH > 7) because of the additional hydroxyl group on the 2' carbon of the pentose sugar (hydrolyzed rapidly, but DNA is not)

Answer 14

cyclic 2',3'-monophosphates

Answer 15

rapidly hydrolyzed further to yield a mixtuer of nucleoside 2'- and 3'-monophoshates

Answer 16

serves a purpose in gene expression DNA is maintained during cell division and during extended periods in nonreplication cells

Answer 17

DNA: resistant RNA: degraded

Answer 18

secondary structure, can fold back on itself to form intramolecular base pairings 3D to fold into many different shapes

Answer 19

RNA base stacking hides the hydrophobic bases away from the hydrophilic surroundings

Answer 20

all the bases are stacked. base-triple interactions, helix-helix packing allows stable 3D folding

Answer 21

1. helical structures 2. internal loops 3. hairpin loops

Answer 22

when the strand folds back onto itself, the paired strands are antiparallel to one another and form a right-handed helix

Answer 23

separation of double-stranded DNA (single stranded RNA that folded back on itself) because lack of base pairing

Answer 24

RNA folds back but there is an unpaired loop of bases at the end of a stem region nucleotides within loops are arranged to maximize hydrogen bonding and base stacking, enhancing thermodynamic stability

Answer 25

hairpin loops

Answer 26

influenced by weak interactions - van der waals stabilize structures

Answer 27

bind to specific sites to help shield the negative charge of thebackbone, allowing RNA to tightly pack together

Answer 28

1. # of GC vs AU base pairs 2. # of base pairs in a stem region 3. # of base pairs in a hairpin loop (more than 10 or less than 5 bases requires more energy) 4. # of unpaired bases - decrease stability

Answer 29

UV absorption - the light absorbed is directly proportional to the amount of proteinnucleic acid present in the sample

Answer 30

purines = 2 rings pyrimidines = 1 ring

Answer 31

alternating single and double bonds that create resonance (partial double-bond character)

Answer 32

pyrimidine = planar purine = nearly planar with a slight pucker

Answer 33

measures the amount of light absorbed by a sample

Answer 34

1. Sample containing nucleic acids is placed inside a chamber and is exposed to ultraviolet light at a wavelength of 260 nm 2. A photo-detector measures how much light passes through the sample – represented on an absorption spectrum

Answer 35

they absorb MORE light. the close interaction of stacked bases when bound within a nucleic acid decreases the absorption of UV light relative to that of a solution with the same conc. of free nucleotides

Answer 36

calculating the concentration of nucleic acids in a solution - the darker a solution, the more concentrated it is as it is absorbing more light

Answer 37

A260 = e260 * c * l A260 = the absorbance of UV light as determined by the spectrophotometer e260 = extinction coefficient c = concentration l = path length / distance that light travels through

Answer 38

the amount of UV light able to pass through a solution greatest at 260 nm - larger for DNA than for protein

Answer 39

dsDNA < ssDNA << free nucleotides

Answer 40

Large decrease in light absorption at 260 nm occurring as single strands of DNA anneal to form double-helical DNA Forces that stabilize the DNA helix like hydrogen bonding and base stacking, limits the amount of resonance that can occur within the aromatic rings of the bases Decrease in UV light absorbed as ssDNA anneals to form dsDNA

Answer 41

Large increase in light absorption at 260 nm that occurs as DNA unwinds/melts As DNA is denatured, the base pairs are disrupted and the 2 strands separate, forming ssDNA The resonance within the bases are no longer constrained, so the UV light absorption of a single-stranded DNA is higher than double-stranded at the same concentration

Answer 42

highly viscious at pH 7 and 25 degrees cels exposed to extreme pH or above 80 degrees viscosity decreases, indicating denaturation

Answer 43

melting and 2 strands break apart - hydrogen bonds and base stacking interactions are disrupted

Answer 44

single-stranded form - the resonance within nitrogenous bases are no longer constrained by the forces that stabilize the DNA double helix

Answer 45

1. track the absorbance of an aqueous solution across a range of temperatures 2. the temp at which half the DNA in the sample is denatured (50% dsDNA and 50% ssDNA)

Answer 46

1. determining base composition 2. analyzing structure and function of DNA (interactions holding the strands together) 3. estimating the amount of G and C bases

Answer 47

higher content of GC = higher melting point (3 hydrogen bonds instead of 2)

Answer 48

T = 0.41 (%G+C) + 69.3 degrees under standard conditions and a salt conc. of 0.2 M

Answer 49

reconstruction of a nucleic acid (or protein) to its original form after denaturation

Answer 50

1. 2 strands must not be completely separated (dozen residues must remain intact) 2. when pH/temp returns to normal, the separated segments of the double helix spontaneously rewind to form an intact duplex

Answer 51

1. Complementary sequences in the 2 strands find each other by random collisions and form a short segment of double helix 2. The remaining unpaired bases come together as base pairs and the 2 strands zip themselves together to form the double helix

Answer 52

2 samples of isolated DNA or RNA are heated to cause denaturation and then are mixed. the sample is left to cool, letting the complementary strands to form a duplex whether it is a DNA-DNA, RNA-DNA, or RNA-RNA hybrid

Answer 53

1. DNA concentration More concentrated sample of DNA has more frequent intermolecular collisions = faster hybridization rate 2. base pair mismatch When hybridizing 2 nucleic acid segments that are completely complementary, mismatched nucleotides can interrupt base pairing, stacking interactions, and DNA stability 3. pH In a range of 5-9, there is only a minor effect on DNA hybridization pH < 5 causes the liberation of all bases from the helix pH > 9 causes the conversion of dsDNA to ssDNA

Answer 54

The extent that hybridization can occur between 2 non-complementary strands of nucleic acid - i.e the degree of complementarity required between 2 strands in order for them to hybridize

Answer 55

Hybridization occurs only when the 2 strands are highly compatible

Answer 56

Hybridization occurs even in the presence of some base mismatches

Answer 57

Duplex formation is not favoured High temperatures – close to melting point Low-salt concentrations Presence of organic solvents

Answer 58

Hybrid formation is favoured Low temperature – below melting point High-salt concentrations Absence of organic solvents

Answer 59

DNA polymerases (enzymes able to synthesize DNA strands using a pre-existing DNA template and free deoxyribonucleotides. They add nucleotides (primers) to pre-existing strands)

Answer 60

2 synthetic primers are prepared, complementary to sequences on opposite strands of the target DNA at positions defining the ends of the segment to the amplified extended by a DNA polymerase

Answer 61

DNA sample containing the segment to be amplified A pair of synthetic oligonucleotide primers Deoxynucleoside triphosphates (dNTPS) DNA polymerase

Answer 62

1. denaturation Briefly heated to separate the strands Enables the components of the reaction to interact with the single-stranded DNA template 2. annealing Cooled so the synthetic primers can anneal to the DNA template High concentration of primers increases the likelihood that they will anneal to each strand before they can reanneal too one another 3. elongation Temperature is slightly increased for synthesis of a complementary DNA strand DNA polymerase recognizes the 3' ends and extends the strands along the targeted segment 4. amplification Steps 1-3 process is repeated for a second time 5. repeat Steps 1-3 are repeated 25-30 times, amplifying the DNA segment between the primers until it can be analyzed or cloned

Answer 63

comprised of short sequences of oligonucleotides that are synthesized commerically

Answer 64

1. 18-25 nucleotides in length 2. 40-60% GC content 3. Annealing temperature in the range of 50-60 degrees Celsius 4. 1 or 2 GC residues at the 3' end of the DNA strands 5. Minimal secondary structure and base repeating 6. Complementary to sequence chosen for amplification

Answer 65

Tm = 2 degrees (A+T) + 4 degrees (G+C)

Answer 66

Ta = Tm - 5 degrees

Answer 67

determine the size of the PCR product

Answer 68

Technique for separating mixtures of large charged molecules like proteins or nucleic acids by causing them to move through a gel matrix when an electric field is applied

Answer 69

1. The sample of interest is added to a slot at one end of the gel. The gel matrix is composed of agarose (a kelp-derived material that does not disrupt nucleic acid base pairing) 2. Voltage is applied to the gel. Since the backbone of nucleic acids are negatively charged, both DNA and RNA will migrate towards the positive end of the gel in the electric field 3. Larger molecules tend to move more slowly than smaller ones, so the molecules are separated based on their size – larger molecules are retained closer to the top and smaller molecules migrate towards the bottom of the cell

Answer 70

used to visualized the PCR product loaded into each well - fluoresces when exposed to UV light, used to detect nucleic acids in a gel

Answer 71

planar molecules = inserts itself in between nucleic acid strands in a nonspecific manner

Answer 72

used to amplify segments of RNA like mRNA gene expression products

Answer 73

RNA is not amplified by PCR, instead it is converted to cDNA, amplified to PCR can amplify and sequence gene segments without introns can quantify mRNA levels as a measure of gene expression using quantitative PCR

Answer 74

SYBR green

Answer 75

a fluorescent label that fluoresces substantially brighter when bound to dsDNA

Answer 76

allows the quantification of dsDNA product inthe reaction after the extension stage of every PCR cycle measures the amount of PCR product present - the more double stranded products produced with each cycle, the more fluorescence detected

Answer 77

1. DNA is denatured at 95 degrees in a solution containing SYBR green 2. Primer annealing (55-65 degrees) 3. Beginning of synthesis 4. Extension (60-72 degrees) 5. Repeated

Answer 78

cycle number at which the threshold is first surpassed - should not be reached without target DNA or cDNA

Answer 79

added in quantitative PCR to control the amplification of non-specific DNA products

Answer 80

SYBR green may bind to non-desired amplification product and produce a florescent signal Non-specific products will melt at a different temperature than the desired PCR product, giving a melt curve with sharp decreases at more than one temperature

Answer 81

dideoxy chain-termination method

Answer 82

method for determining the nucleotide sequence of DNA

Answer 83

DNA polymerases (to mock the mechanism of DNA synthesis - the 3'-hydroxyl group of the primer reacts with an incoming nucleotide to form a new phosphodiester bond ) labelled primer and dideoxynucleotides (for enzymatic synthesis of a DNA strand complementary to the strand under analysis)

Answer 84

chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing

Answer 85

ddNTPS (have H at both 2' and 3' positions) interrupt DNA synthesis because they lack the 3'-hydroxyl group needed for the next step

Answer 86

1. DNA Denaturation - Apply heat to DNA sample causing the dsDNA to ssDNA, forming template and complementary strands 2. Primer - DNA primer is annealed to the template strand, allowing nucleotides to be added later - Primer is added because DNA polymerase requires a free 3' hydroxyl group to add free nucleotides - Primer was radiolabeled in original Sanger sequencing, which allowed the products of the DNA synthesis to be detected by an autoradiogram 3. Free Nucleotides (dNTPS) - 4 reaction mixtures are set up and the template strand (with primer) is added to each one along with DNA polymerase and free nucleotides 4. Modified Nucleotides (ddNTPS) - Used to terminate the synthesis reaction (no 3'-hydroxyl group, has only –H) - ddNTPS are added to each reaction mixture - Only 1 type of ddNTP is added to each reaction mixture, ddATP (green), ddTTP (red), ddCTP (blue), or ddGTP (yellow) - Added at much lower concentration than dNTPS, allowing for the extension of the synthesized strand with unmodified dNTPS before a ddNTP is added to terminate the extension 5. Chain Termination - ddNTPS lack a 3'-OH group required for the formation of a phosphodiester bond between 2 nucleotides - Causes DNA polymerase to cease extension of DNA 6. Gel Electrophoresis - Sample is collected from reaction mixtures - DNA is separated by size using gel electrophoresis - Each reaction mixture is added to a separate lane of the gel; the radiolabeled primer is detected by autoradiography - All possible chain lengths produced are separated by 1 nucleotide - Shorter fragments run further on the gel than the longer ones

Answer 87

1. All ddNTPS are added to the same reactin (blue, green, yellow and red) 2. Products of different size are separated by size using capillary electrophoresis The fluorescently labeled segments are excited by a laser and the wavelength of the fluorescent emission (red, green, yellow or blue) is detected, one nucleotide at a time

Answer 88

1. all ddNTPS are added to the same reactin (blue, green, yellow, red) 2. Products of different size are separated by size using capillary electrophoresis. The fluorescently labeled segments are excited by a laser and the wavelength of the fluorescent emission (red, green, yellow or blue) is detected, one nucleotide at a time

Answer 89

For DNA segments flanked by regions with known sequences, PCR primers can be designed to amplify the region of interest If the sequence is unknown, synthetic adapters with known sequences can be ligated (enzyme joining 2 nucleic acid fragments) to the ends to serve as primer binding regions, enabling PCR amplification

Answer 90

Segment is incorporated into a vector by a process called molecular cloning Once inserted into bacteria, the vector can be replicated as the bacteria proliferate, enabling amplification of the DNA segment of interest

Answer 91

method to provide large quantities of purified DNA for sequencing

Answer 92

Isolation and generation of recombinant DNA molecules that are placed in organisms for replication and study Separates a specific gene or DNA segment from a larger chromosome and incorporating it into a small molecule of carrier DNA modified DNA is introduced into a host cell and replicated

Answer 93

1. Obtaining the DNA segment to be cloned - Enzymes called restriction endonucleases act as precise molecular scissors, recognizing specific sequences in DNA and cleaving genomic DNA into smaller fragments suitable for cloning - Alternatively, genomic DNA can be sheared randomly into fragments of a desired size - If the sequence of targeted genomic regions is known, some DNA segments that will be cloned are simply synthesized 2. Selecting an appropriate carrier molecule of DNA capable of self-replication - Called cloning vectors and act as carriers of new DNA - Ex: plasmid vectors and bacterial artificial chromosomes 3. Joining 2 DNA fragments covalently - The enzyme DNA ligase links the cloning vector to the DNA fragment to be cloned (inserted) - Composite DNA molecules of this type, comprising covalently linked segments from 2 or more sources are called recombinant DNA 4. Moving recombinant DNA from the test tube to a host organism - The host organism provides the enzymatic machinery for DNA replication - Bacteria are often used for this purpose 5. Selecting or identifying host cells that contain recombinant DNA - Cloning vectors have features that allow the host cells to survive in an environment where cells lacking the vector would die (ex: antibiotic resistance) - Cells containing the vector are selectable in that environment

Answer 94

DNA molecule known to replicate autonomously (self-replicate) in a host

Answer 95

a segment of DNA is placed within a cloning vector, allowing for its replication

Answer 96

circular DNA molecule found in bacteria that replicates separately from the bacterial chromosome

Answer 97

when cloning fragments are less than 15,000 bp in length

Answer 98

Plasmids have specialized sequences that enable them to use the cell's resources for their own replication and gene expression to survive Naturally occurring plasmids have a symbiotic role in the cells (ex: performing new functions for the cell, resistance to antibiotics, etc)

Answer 99

1. Ori (Origin of replication) - Sequence where replication is initiated by cellular enzymes - Required to propagate the plasmid 2. Restriction Sequences - Several unique restriction sequences are targets for restriction endonucleases - Providing sites where the plasmid can be cut to insert foreign DNA 3. Number of Base Pairs - Facilitates both its entry into cells and the biochemical manipulation of the DNA - Generated by trimming away many DNA segments from a larger parent plasmid (sequences that the molecular biologist does not need) 4. Antibiotic Resistance - Have genes that confer resistance to antibiotics tetracycline and ampicillin - Allows the selection of cells that contain the intact plasmid or a recombinant version of the plasmid using these antibiotics

Answer 100

1. plasmid generation 2. transformation and antibiotic selection

Answer 101

1. The pBR322 plasmid DNA is cleaved at the restriction site by a restriction endonuclease called Pstl. The foreign DNA contains the Pstl complementary ends 2. Foreign DNA fragments are ligated into the plasmid. Successful integration disrupts the AmpR gene, making the plasmids no longer resistance to ampicillin

Answer 102

1. Plasmid DNA is introduced into the bacterial cells by transformation, whereby the cells and plasmids are incubated at 0 degrees in a calcium chloride solution and then heat shocked by raising the temperature to 43 degrees (chemical transformation). Alternatively, the cells can be subjected to a high-voltage pulse to allow the plasmid DNA to enter (electroporation transformation) 2. The cells are grown on agar plates with tetracycline to select only for those that have taken up the plasmid. Individual colonies are transferred to matching positions on additional plates. 1 plate has tetracycline and the other has tetracycline and ampicillin 3. Cells that grow only on the tetracycline agar plates contain recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA. Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates mean that cloning was unsuccessful

Answer 103

Have origin of replication, antibiotic resistance, restriction sites and a reporter gene (enables the detection or measurement of gene expression) BACs have stable origins of replication to support very long segments of cloned DNA

Answer 104

Slow and expensive Read lengths are only up to 1000-1500 bases Sequences for a large segment of DNA need to be broken down, analyzed one at a time and compiled together

Answer 105

rapid sequencing of large DNA segments

Answer 106

1. These large DNA segments (could be an entire human genome) are fragmented to smaller segments (300-400 bp) and are sequenced simultaneously 2. The sequences for overlapping fragments are then aligned to generate a consensus sequence for the entire DNA segment

Answer 107

modified nucleotides bearing a reversible terminator (RT)

Answer 108

sanger = labeled with fluorescent tags RTS = the fluorescent tags are bound to the modified nucleotides using a cleavable linker region

Answer 109

1. library preparation 2. cluster generation 3. sequencing 4. data analysis

Answer 110

DNA fragmentation: - Large DNA segments are cleaved into 300-400 base pair fragments - Fragmented ends are repaired and a single adenosine nucleotide is added to the 3' ends of the fragments to prevent them from ligating to each other adaptor ligation: - Ligated to the 5' and 3' ends of the fragmented DNA segment - Terminal sequences = essential to the next stage of sequencing, known as Cluster Generation - Index sequences = allow DNA libraries from different samples to be processed and separately and pooled together in the same run - Each index is like a unique barcode for DNA fragments from a specific sample. After the run, the sequences from individual samples can be separated from each other based on this indexing barcode - Primer binding sequences = serve as binding regions for sequencing primers - Allows for paired sequencing from both ends of the DNA fragment

Answer 111

Involves the amplification of individual sequences from the DNA library to form clusters of clonal (all from the same gene sequence) DNA segments Takes place in the flow cell, a piece of acrylamide-coated glass that is coated with oligonucleotides

Answer 112

1. DNA library is added to the flow cell. terminal sequences in the adapters allow single DNA segments to hybridize with the oligonucleotides bound to the surface of the flow cell. a DNA polymerase is used to extend an oligonucleotide that is complementary to the bound DNA molecule 2. the original template is washed away, leaving only the newly synthesized strand that is covalently bound to the flow cell 3. the adapter sequence at the 3' end of the bound DNA molecules hybridizes with a nearby oligonucleotide. the bridge is extended and the 2 strands are denatured 4. process is repeated, forming a cluster of forward and reverse strands - reverse strands are hydrolyzed and washed away, leaving a cluster of unidirectional clonal strands

Answer 113

3 components added to the flow cell: 1. 4 fluorescently labeled reversible terminator nucleotides (RT-dATP, RT-dCTP, RT-dGTP, RT-dTTP) 2. a sequencing primer that can hybridize with the 3' adapter region of the bound DNA segment 3. DNA polymerase

Answer 114

graph with many coloured dots - each dot represents a stage of amplification and the colour determines the next nucleotide (e.g, green cluster = next nucleotide is A)

Answer 115

aligned to a reference genome once all the reads are generated "depth" of coverage = # of times that a specific base pair appears in a sequence read

Module 3 Sections 1-5 Flashcards

Section 1: DNA Structure and Function Section 2: RNA Structure and Function Section 3: Quantification of Nucleic Acids Section 4: Amplification of DNA by PCR Section 5: Methods for DNA Sequencing (140 cards)