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Quiz 1 - Bio14a (DNA Replication + PCR, DNA Seq, Sanger)
Terms in this set (98)
DNA double helix structure
1. ANTIPARALEL = Phosphodiester bonds on the outside - polar and negatively charged (5'-3', 3'-5), This is why one strand is always replicated backwards
2. COMPLIMENTARITY = Bases on the inside
a. H BONDS Interact noncovelently through base-paring and
b. SPECIFC: C triple bond with G, A=T, groups of H donors and H acceptors
3. Minor Grove: 2 backbones are closer together
4. Major grove: 2 backbones are farther apart
- A and T are equally abundant in any given double-stranded DNA; likewise G and C are equally abundant in any dsDNA
1' C = where N base attatches
2' C = OH attatched to it in DNA, H in RNA
3' C = forms phosphodiester bond
5' C = outside the ring strong, where P group attatches
The variable part of DNA? What makes DNA sequences different?
- the phosphodiester backbone is always the same
- the purine and pyramidine order of bases are what make it different
Similarity between U and T bases
- Both have H-bond donors and acceptors in the same places so they can both base-pair with A
Are the bonds between paired bases covalent or noncovalent?
- Base pairing is mediated by noncovalent hydrogen bonds.
It is reversible: the strands can separate and come back together without chemical change
Functions of DNA, RNA, and protiens and matching
1. Proteins -- Amino acids
- do work in the cell
2. Nucleic acids --Nucelotides
DNA - stores genetic info in a cell
RNA - stores and is used as coding material
3. Carbs - sugars
4. Lipids - fatty acids
Differences between DNA and RNA
1. Both have negative phosphodiester bonds, (5'-3")polarity
2. Different Sugars... DNA: Missing an O at the 2', RNA has an O at the 2'
3. Different N base... DNA: has a T base RNA: has a U base
4. Different shape ... DNA: double helix RNA: ssDNA
Rank IMF by strength and reversibility
- covalent bond
- ionic bonds
- hydrophobic interactions
Hydrophobic interactions < hydrogen bonding < ionic bonding < covalent bonding
- a pair of valence electrons that is shared between two nuclei
- the strongest bonds
- functional unit region of DNA (or RNA in viruses)
- provides instructions to make 1+ gene products
- RNA and proteins
- created by DNA
serve as templates for other molecules
DNA -- mRNA
RNA -- protein
don't fall into gene product categories
For a cell to divide, what has to happen
- DNA must be copied
1. DNA is packaged in chromatin
2. Chromosomes condense, line up, seperate, into sister chromatids
- product of replication of same starting molecule
DNA is wrapped around histone octamers to form nucleosomes, which are then further folded up into tightly-wrapped, compact structure.
Two of the main ingredients in most plant fertilizers are phosphorus and nitrogen. Which
classes of biological molecules in the plants are the nitrogen and phosphorus incorporated
Phosphorus - Nucleic acids (DNA/RNA phosphate backbone)
Nitrogen - Both nucleic acids (N bases) and proteins (NH2).
what is the term for a pair of electrons shared between two nuclei?
TWO shared pairs of electrons
covalent double bond
Make a cells genes?:
No cell's genome is RNA. Viruses aren't cells
RNA and protein
Draw out the chmical structure of dTTP.
- Identify the deoxyribose group
- Phosphate groups
- Nitrogenous base
- Notice last phosphate has an OH bounded to it
- Deoxyribose group is the 5C sugar ring, 4 Carbons in the ring, 5 is outside the ring
- Nitrogenous base
- Each solid line represents one covalent bond. (There are some H not drawn). C makes 4 covalent bonds, so assume hydrogen.
- between H (donor) and highly electronegative atom (acceptor)
- Hydrogen = H bond donor
- Electronegative atom = H bond acceptor
- water is both donor and acceptor
- easily broken by heat
DNA exists as a double helix (two long molecular chains held together along their entire
length). As you may know from movies like Jurassic Park or TV shows like CSI, DNA is an
extremely stable molecule. However, cells routinely separate the DNA double helix to express
the genetic code or to copy it. What aspect of the double-helix structure allows DNA strands
to be separable, but also stable?
seperateable - noncovalent bonds - Hbonds, ionic bonds
In this particular case, the interaction
between DNA strands is through hydrogen bonds: 2 to 3 of them per basepair. They make the pairing
of complementary DNA strands highly specific, but not irreversible.
The strands can be separated by
high temperature, and can then re-anneal (come back together) at a lower temperature. When they
re-anneal, the complementary base-pairs will re-form their hydrogen bonds
Brca1p is a protein which is normally involved in protecting cells from the harmful effects of
DNA damage. What is the molecular make-up of the BRCA1gene, and how does it
contribute to production of the Brca1 protein?
- gene is composed of DNA, on chromosome
- DNA sequence is used to make BRCA1 mRNA via transcription
- BRCA mRNA serves as a template and production of protein via translation
- DNA sequence of the gene indirectly determines the proteins amino acid sequence
Draw a simple digram of DNA replication bubble
- label 3'--5'
- mark ORI with stars
- leading strand primer arrow
Every leading strand in the diagram should have one primer. A proper replication bubble has 2
leading strands, one for each replication fork.
No lagging strands in the diagram should have primers on them. If they do, we cannot tell whether
you can distinguish leading and lagging strands
Define DNA polymerase. What type of molecule is a DNA polymerase? Is it a gene product?
What function(s) do all DNA polymerases have? What additional functions, if any, do some
DNA polymerases also have?
1. DNA polymerase is a protein (enzyme)
2. All proteins are gene products
3. Adds dNTP to the 3'OH end, elongates DNA,
4. 3' OH AND Primer is needed to for DNA polymerase to add dNTPs (create phosphodiester bonds)
5. DNA polyemerase have exonucleases - proofreading mechanism that breaks phosphodiester bonds if necessary
Add to the adedinine base, the chemical structure of dATP
- Adenine attaches at 1' C
- Deoxyribose sugar, missing O on 2' C
- 3 phosphate groups on 5'C
- Remember the OH at the end and the charges
How can you tell if whether one is the template strand or the nascent growing strand?
- growing nascent strand has a free 3' OH which is ready to accept a new nucleotide opposite the C in the template strand.
- You can tell the nascent molecule is not RNA, because it does not have a 2' hydroxyl, so you know
this is not transcription. Translation is not nucleic acid synthesis.
Proteins involved in DNA Repliciation (6)
1. Topoisomerase - relives the stress of supercoils
2. DNA helicase - unzips DNA (H-bonds) at rep. forks
3. SSBPs - stabilizes ssDNA
4. RNA primase - lays down RNA primer bp with template
5. DNA polymerase - extends RNA primer adds DNA nucleotides and has exonuclease activity
6. DNA ligase - connects DNA fragments
1. ssDNA is unstable
2. No ssDNA template
3. DNA polymerase cannot syntehsize DNA without a 3'OH to extend from/DNA polymerase cannot join 2 free dNTPS
4. There is no 3' to 5' DNA polymerase
5. Lagging strand has nicks
6. DNA polymerase occasionally inserts an incorrect nuceleotide
1. SSBP - bind to and protect ssDNA
2. Helicase - seperates the two strands (H-bonds) of the double helix at the replication fork
3. Primase - lays down RNA primer
4. Lagging strand synthesis: discontinous synthtesis of one strand (involving repeated primer synthesis) and ligation of resulting Okazagi framents)
5. DNA ligase catalyzes phosphodiester bond formation to seal nicks
6. Exonuclease proofreading activity of polymerase removes errors
1. semiconservative - 1 new, 1 old
2. Produces identical sister chromatids
3. Each parental strand serves as a template for the syntehsizes of a daughter strand
4. DNA polymerase catalyzes phosphodiester bond, one dNTP at a time
5. Incoming dNTP is always added to the 3' end of the nascent strand!
6. Because DNA is antiparallel, one strand is replicated backwards (lagging strand, leading stra)
DNA Template important
- NOT an enzyme or catalyst used to make the product
- It is the provider of information, provides structure for the growing nascent strand
are phosphodiester bonds broken
phosphodiester bonds of older strands are not broken, but strands are separated to basepairing with the nascent (being-made) DNA
baseparing in antiparallel direction
Relationship between DNA, chromosomes, chromatids, and chromatin
Lecture 3 Goals
Apply your knowledge that nucleic acid strands always anneal in an antiparallel configuration.
Apply the rules of base complementarity in a variety of contexts
Explain the relationships between DNA, chromosomes, chromatids, and chromatin
Explain difference between sister chromatids and non-sister chromatids
Hydrogen bond donor
- covalently bonded to a hydrogen;
- this atom must be one of the more electronegative atoms (N, O, S)
- has a lone pair of electrons. The result of this is that the hydrogen has a partial positive charge.
hydrogen bond acceptor"
- electronegative atom with a lone electron pair, having a partial negative charge
hydrogen bonding occurs
A hydrogen bond is the pairwise interaction between one acceptor and one donor. The two electronegative atoms "share" the hydrogen nucleus between them, and are brought into closer proximity than they would otherwise be.
N, O, S, NOT C
- because of the polarity of water molecules
.- water avoids interaction with nonpolar molecular wants to satisfy partial charges, too high of energy cost
- cannot interact favorably with the partial charges in water molecules.
DNA replication Eukaryotes
- Multiple origins of replication to compensate for long linear chromosomes
- Replication bubble
The other strand, called the lagging strand, has its 3' end pointed away from the replication fork, and so is synthesized in short, discontinuous pieces.
This is because the polymerase can only make DNA in one direction: Adding a dNTP on to the 3' hydroxyl of the growing strand.
How many DNA polymerases need to be at the replication fork?
Two are engaged in DNA synthesis at each fork: one on leading strand and one on lagging
DNA replication basic steps
1. Helicase separates strands
2. Primase synthesizes RNA primer
3. DNA polymerase synthesizes new DNA
4. RNA primer(s) are removed and replaced with DNA
5. DNA ligase seals nicks in phosphodiester backbone
origin of replication, which is a particular DNA sequence recognized by specific DNA-binding proteins that recruit all the other proteins needed for DNA replication.
activity, ability to cut the outside nucleotide off cut phosphodiester bond
Electrophoresis: assesses mass to charge ratio of substance
Result: High mobility (characteristic of DNA bc of mass to charge ratio)
1. Electric current in DNA
2. DNA runs towards positive electrode, runs through gel of complex fibers, big molecules get slowed down, small molecules move more quickly.
3. Small molecules are lower in the wells bigger molecules at the top
- Agarose gel- usually used to separate DNAs based on size/shape. LARGE molecules
- Acrylamide gel is good at sperating small molecules (proteins)
- determines DNA nucleotides
- wanna see the sequence of DNA and how they work
- compare the differenct sequences
between humans = single nucleotide polymorphism
-- amplifies region of DNA template
- useful for visualization gel electrophoresis and sequencing
- three cycles: Denautration (95°C), Annealing (60°C), and Elongation (70°C)
- 2^n copies
- get one product just many copies of it, if you get multiple bands, means your primer is annealing more than once
PCR three cycles
1. Denature - 95C for 30s-60s so that H-bonds break
2. Annealing - for 30s* depends on primer length, longer primer = higher melting temperature LOW T = imperfect sequenc specificity, High T = too specific primer will not bind. We can estimate the primer length from the what the T melt should be
3. Elongation (70°C) - depends on polymerase, 1 min/1kb
PCR ingredients (5)
1. DNA ds Temlate - tiny bit
2. DNA polyemerase Taq - evolved to withstand heat
4. 2 primers - ssDNA18-20 nt
- designed by experimenter
- enough to be specific not to much to anneal to themselves
- complementary to the ends of the region of template DNA being amplified
issues with proteins and gel electrophoresis
The SDS (detergent) is essential for coating the proteins with negative charges so that they will run from negative toward positive electrode. Unlike DNA, proteins really do have a lot of variation in Mass-to-charge ratio. The SDS drowns out these differences.
agarose gel example
Where are the biggest DNA molecules?
Where are the smallest?
Where are the most abundant DNA molecules?
Where are the least abundant DNA molecules?
Does staining allow us to see all the DNA?
- Determine exact base sequence of DNA
- use DNA synthesis with chain-terminating nucleotides ddNTPS, lack OH at the 3' end. We can't add DNA nucleotides at this end.
- Create DNA products of every length and know the identity of the last base added for each product
- Make DNA copies that are terminating early at every different nucleotide
Each band on a gel
- represents many molecules of a significant concentration
does shape influence how things run on a gel?
- shape does influence how molecules run in a gel, but its effects are complicated. For this reason, when we analyze proteins (which can have very different shapes) by gel electrophoresis, we usually "denature" the proteins first, so that they will all be of similar shape.
Both PCR and cellular DNA replication involve _____.
b. dNTPs (specifically, dATP, dCTP, cTTP, dGTP)
c. DNA primers which are made before the DNA synthesis reaction occurs.
d. NTPs (specifically, ATP, CTP, UTP, GTP)
e. Helicase to unwind the DNA
True of cellular DNA replication, but PCR uses heat to melt apart DNA strands.
f. RNA primers which are made on the template strand during DNA synthesis.
g. A DNA polymerase enzyme.
b. dNTPs (specifically, dATP, dCTP, cTTP, dGTP)
g. A DNA polymerase enzyme.
When DNA is replicated, which part of the chain is a new dNTP added on to?
a. The nitrogenous base at the 3' end of the strand
b. The 3' hydroxyl (OH)
c. The nitrogenous base at the 5' end of the strand, which is complementary to the incoming base
d. The 5' phosphate (PO4-)
b. 3' OH
Which of the following functions are served by a set of primers used in PCR (polymerase chain reaction)? Choose one or more (all that apply).
a. Anneal (hybridize) to specific, complementary region(s) of the DNA template(s)
b. Define the boundaries of the region which will be amplified by PCR.
c. Be incorporated into the final product, along with dNTPs
d. Initiate replication of both strands of the DNA templa
all of them
Which statement(s) below accurately describe separation of molecules by gel electrophoresis? Choose one or more:
Select one or more:
a. Larger DNA molecules move more quickly through the matrix than smaller ones, because the large molecules have more negative charge.
b. Larger DNA molecules move more slowly through the matrix than smaller ones, because of their size.
c. Each band visualized on a gel represents one single molecule.
d. During normal gel electrophoresis, any given molecule will move down the lane it was loaded in, and will not migrate from one lane to another.
e. A circular and a linear DNA molecule, if the same size, will move through the gel at the same speed, because their shape does not affect how they travel through the matrix of the gel.
f. Gel electrophoresis can be used to analyze proteins, DNAs, or RNAs.
b. d. f.
1. ss comp. (DNA/RNA) anneal to other ss nucleotide, forming a double helix. Anneal via complemenatary base pairing
2. Specific -- Control specificity by t. High T - perfect complementarity, very few come together Low T - imperfectly matched bp comes together.
3. Reversible -- because we can melt them apart and put them together
4. EX: Southern (DNA) and Northern (RNA) Assays finds a specific sequence
Southern Blot basic steps
There is a known viral oncogene gene - HPV (probe). Do vertebrate genomes (sample) contain it?
Start: Genome - Total DNA - would show up as a big smear
1. Digest with restriction enzymes
2. Load DNA samples to gel - along DNA ladder to compare size.
3. Run gel electrophoresis - seperates by size. Small molecules go faster towarsd bottom
4. Transfered/Blotted to filter paper - fixed and accessible for probe to attatch
5. Incubate blot with probe - ssDNA 100 bp long (longer than primer), comp., labeled radioactively or fluorecently
6. Wash away excess probe - renatured DNA stays in place, probe goes out
7. Detect location of probe Band = where probe sticks to complementary sequences = gene found. Indicates size and number of copies of DNA sequence present in the starting sample
determine whether sample included a
DNA sequence complementary to the probe sequence, and what size restriction
fragment(s) complementary sequences are found in.
- ssDNA 100 bp long (longer than primer), comp. or mostly comp.
- anneals to sample genome in southern or nothern (contains viral gene) and labeled
- can be obtaned by chemical isolation of DNA gragment, PCR, or nucleotide seque
The differences between Southern and Nothern
1. Sample Genome would be RNA not DNA
2. No need for restriction enzymes because its short
Everything else is the same
Hybridization basic steps
- must be complementary or they will not anneal
1. Start with ds DNA or ss RNA
2. If DNA - -seperate with heat to make ss
3. Attatch probe
4. Probe attatches you id the complimentary sequences and can use that to hybridize
- cut only only DNA (RNA is not necessary since its short already)
- recgonizes short 4-6 nucleotide sequences (restriction site) in ds DNA and cleaves DNA at these sites
- palindomic - read the same on opposite strands
- Since restriction enzymes are specific, DNA from the same species will yield fragments of similar length.
- Gel seperates DNA fragemnts according to size in kb in thousands of bp
what would result in a high melting T
- if there are more hydrogen bonds that can form between the two single strands of nucleic acid!
- Higher GC content (corresponding lower AT content)
- Longer sequence
- The higher the Tm for a hybrid molecule, the easier it is to anneal its two strands together.
- Determine the base sequence of a specific piece of DNA using ddNTPs
- Create DNA products of every length, terminating of every other nucleotide, and knowing the last nucleotide added to every product
-chain-terminating nucleotides are dideoxynucleotides, which lack OH at the 3' end. We can't add DNA nucleotides at this end.
- Tagging the ddNTPs each with a different colored dye allows you to run ONE lane of gel instead of four!
- After they are seperated by size, a trance of the flurecence as diff colored peaks is read on graph. THe diff color peaks represent the order of dlurescently tagged DNA nucleotides of the daughter strand
PCR vs. REgular cell replication
1. BOTH: cell replication and PCR use dNTPs and DNA polymerase
2. DIFF: PCR uses DNA primer while cell replication uses RNA primer, more unstable
Sanger vs. PCR Ingredients (4 things)
1. DIFF: DNA ds Temlate - LOTS of it
2. DIFF: 1 primer vs. 2 primers (PCR)
3. DIFF: sanger uses ddNTPs
4. SAME: DNA polyemerase, dNTPS, and buffer
Sanger vs. PCR Implifications
1. Sanger: Linear Amplification
- copies only one strand
- copy is the same as the direction of the primer and cannot be used as template later
- linear amplification - not exp, so we lots of it
2. Sanger: ddNTPs in addition to dNTPS
3. Both use gel electrophoresis to seperate DNA fragments according to size
4. same 3 cycles tho
Sanger sequencing basics
1. Ingredients (6) DNA poly, dNTP, buffer, dsDNA, primer, ddNTPs (labeled), put in a tube and.
2. DNA polymerase starts at strand and makes a full length copy
3. ddNTP is aded - replicates DNA fragments at diff lengths
4. Run on a gel to see where DNA is and size
5. We can find the order on a graph
- Melt apart double stranded DNA to be sequenced
- Primer anneals at specific site on template DNA to be sequenced
- DNA polymerase extends primer, adding dNTPs to its 3' end
4. Addition of ddNTP
- DNA polymerase eventually incorporates a ddNTP, stopping synthesis at a random point
- The same region of the DNA is partially copied like this many times over, producing many copies of each length of product, and many different lengths of products
5. Gel electrophoresis
- Sample is loaded into gel
- Electric field is applied, pulling DNA toward the positive electrode
- DNA separates into bands of distinct sizes (each of which is a population of molecules of the same length)
6. Analysis: Visualize the bands on the gel
- Distance from well of each band shows you its length in nucleotides
- Final (3') base identity is shown either by which lane band is found in (as in example above) or by color of band (when fluroescently tagged ddNTPs are used)
Sequencing - temination of growing strand
CHAIN TERMINATION IS PREMATURE TERMINATION OF THE NASCENT STRAND, NOT DIGESTION OF PRE-EXISTING DNA
Goals for Lecture 4: Identify molecular interaction partners of key players in biological processes, such as: DNA polymerase, primase, helicase, ligase, template DNA, dNTPs
Understand the steps of each biological process (in this case, DNA replication): what order steps occur in, function of each step
Understand the steps of each method (in this case, gel electrophoresis, Sanger sequencing): what order are they done in, and what is the function of each step.
Predict the outcome of an experiment properly performed (in this case: gel electrophoresis or Sanger sequencing).
Interpret actual experimental results (in this case: gel electrophoresis, Sanger sequencing)
1. uses DNA molecules from two sources
donor DNA - dsDNA fragment of interest, one that you want to express in vector
vector DNA - where donor DNA is inserted, plasmid
2. Both DNAs are cut with restriction enzyme - have the same overhangs, complementary
3. The ressulting framents are mixed together via DNA ligase
4. The recombinant protein is introduced into a bacteria cell via transformation - take up DNA from the outside
5. As bacteria replicates and divides the recombinant DNA also replicates and divides
what is necessary for designing primers?
Primers must be at each end of the sequence. One comes from each strand.
Both should have their 3' ends "pointing" toward the region to be amplifieB
You carry out your PCR reaction using particular temperatures for the steps of:
annealing, denaturation and extension. Which of these steps requires the
Denaturation - unwinding LONG DNA
Annealing is cooler because you are just bp with primer (18-20nt) in length
Low T at annealing
allows for nonspecific bp visualiztion of multiple bandsa
High T at annealing
- too specific, could produce no product or less product
Low denaturation T
- template doesn't sep because Hbonds dont, primers cant bind, no product
Low elongation T
- primer binds but DNA polymerase cannot add dNTPs so no product or few product
what can account for low product
- High T annealing
- Low T denaturation
- Low T elongation
will one molecule of 9000kb show on gel electrophoresis
Imagine you were setting up Sanger sequencing reaction, but you forgot to
add any dideoxynucleotide to the mix. If you ran the reaction product out on a gel like
the one above, how many bands of DNA would you see?
ss nucleotide to anneal to another ss
DNA or RNA of complementary sequence. This involves the formation of a double helix,
stabilized by hydrogen bonds between complementary bases and by base stacking (involving van
der Waals interactions) between adjacent layers of basepair
difference is shape and chemistry in southern
The differences in shape and chemistry that result from sequence differences are used in the
restriction digest step, when the restriction enzymes bind to specific base sequences and cut
the DNA backbone.
point mutation vs. polymorphism
Point mutation - occurred right now. Polymorphism is something that is common in the population, variation in the population . For it to be common, the mutation happened a long time ago
Unexpectedly, you also find that in some of the men you try to genotype, no
bands are detected by Southern blot. Describe one genetic difference between the X
chromosomes of these men and other individuals that could account for this
There would be some mutation that prevents the binding of the probe to the DNA; e.g., a small
deletion or other DNA sequence change in the region complementary to the probe.
Changes in the restriction sites would not result in no band, but would just change the size of the
Each structure is one double-stranded DNA molecule, wrapped around proteins
A woman's doctor tells her to gargle with salt water. She stirs a tablespoon of salt into warm water, and watches it dissolve. Why does the salt dissolve in water?
The partially-negative oxygen atoms in water are attracted to the chloride ions.
d. The partially-positive atoms in water are attracted to the sodium ions.
Nucleic acids perform which of the following functions in living cells?
1. Catalyze chemical reactions (RNA)
2. Provide information required to make specific DNAs (transcription, reverse transcription)
3. Storage of genetic information Correct
4. Provide information required to make specific RNAs
5. Regulate gene expression
6. Provide information required to make specific proteins
hydrogen bond acceptors
O, N, O (of water)
hydrogen bond donors
HN, HO, HOH
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