Exam 2 Flashcards
Explain DNA as an information carrier.
-Hydrogen bonding between bases A-T and G-C allows for the production of exact copies of encoded information and this can be replicated
-GTAACGC on one stand means CATTGCG on the other strand
The discovery of DNA suggested a mechanism for the transmission of genetic information
What grooves does DNA form and why do they matter?
Major and minor
-these are sites of interactions with protein and nucleic acids
What is the most common helix form of DNA?
Right-handed helix
what is the directionality of the DNA double helix?
Antiparallel strands: Opposite directionality
How many base pairs make up the most common Helix?
Most common B has a range of 10.1-10.5 bp/turn
roughly 10
What are other forms of DNA?
Describe the direction, occurrence, and condition
B-DNA: Right, Most common, normal
A-DNA(11bp/turn): Right, RNA-RNA, and RNA-DNA, If water is removed, B to A change
Z-DNA(12bp/turn): Left, near transcription start sites, alternating GC’s
What is a palindrome?
DNA where two complementary strands have the same sequence when read in the 5’3 direction or the 3’-5’ direction
ex.
5’<- GAATTCGAATTC-> 3’
3’ <- CTTAAGCTTAAG-> 5’
What is an inverted repeat?
-Complementary sequence that occurs on the same strand of DNA or RNA but in the inverse direction
-Allow for the formation of hairpin or cruciform structures
What is a mirror repeat?
-Inverted repeat sequence is nonpalindromic
What is the biological significance of different DNA structures?
-Slowing or blocking protein synthesis by the ribosome attenuation seen in prokaryotic-specific
-recognitions sites for restriction enzymes
-recombination of DNA ( genetic information on two chromosomes is exchanged seen in meiosis)
-regulation of gene expression
primary structure different from DNA and RNA
Uracil rather than Thymine
-2’OH on the sugar
-this makes RNA more labile/sensitive to hydrolysis, especially in an alkaline solution
-important for RNA mediate catalysis (splicing)
-Allows or additional hydrogen bonding between segment of RNA
When and how was the structure of tRNA discovered?
in the 1970s with the use of X-ray crystallography
RNA is generally single stranded name the an exception
in dsRNA genomes of some virus
RNA does not always adopt a specific 3-D structure
True
What can happen to the covalent back bond of DNA and RNA naturally?
slow nonenzymatic hydrolysis of the phosphodiester bonds
Who is most readily hydrolyzed under alkaline(basic) conditions in a test tube
RNA
What happens when a single-stranded RNA folds upon itself?
-It can form short-base paired or partially-base paired segments connected by unpaired regions
-Mostly single stranded but can be double-stranded as well ( when ds also right-handed helix known as A form)
—-you can have RNA-RNA hybrids
—-you can have RNA-DNA hybrids
What are examples of NON-watson crick base pairing in RNA?
Two Adenine coming together
Guanine and Uracil getting together
How are we able to measure DNA?
DNA can absorb UV
-Nitrogenous bases are aromatic
-bases absorb UV light near 260nm
-UV absorbance is used as. method for detecting nucleic acid using a spectrophotometer
How do you break the phosphodiester bond backbone?(DNA or RNA)
- this could be done via enzymes like restriction enzymes ( bacteria were identified with the enzymes that allow the recognition of specific sequences and cut the portion into pieces)
-Or a rare occurrence of spontaneous breaking
How do you break the duplex of DNA or RNA?
It’s like opening a zipper by breaking hydrogen bonds
-through the use of heat
-using enzymes like helicase enzymes that unwind the helix in DNA replication example.
Describe the denaturation of double-helical DNA and RNA
-dsRNA solutions are highly viscous at pH 7 and room temperature at 25 Celsius
-When these solutions are subject to high temperatures (above 80 C), viscosity decreases indicating that the DNA has undergone a physical change
* Disruption of H bonds and the base stacking
* DNA denatures when heated slowly
Tm
denaturing/melting
*pH changes can play a role as well as temperature
-the tempature where half of DNA is no longer double-stranded hence the denaturing
Explain the relationship between DNA and Tm
*Each DNA duplex has a characteristic
denaturation temperature or melting
point Tm
* The higher the CG content, the higher the Tm
* If you hold pH and ionic strength constant, you can use Tm to determine base composition
Describe the interaction of DNA with UV light when experiencing changes
- Close interaction of stacked bases in a dsDNA, has the effect of decreasing its absorption of UV light
- UV light absorption of single-stranded DNA is higher than dsDNA.
- The transition from dsDNA to ssDNA can be followed using UV
What is renaturation?
*When the temperature returns to normal,
spontaneous rewinding
* This process is called annealing(Ta): reformation of all the base pairs in the double helix
* At first, strands find each other by random collisions
* Finally, the remaining bases come together as base pairs
What is an extension?
The copying of DNA
In controlled conditions which base of complimentary bases will denature first?
AT
Why is strand separation crucial?
DNA replication, Transcription
Describe the trends of melting points seen in DNA and RNA
- DNA rich in GC versus AT has a higher melting
point - RNA-RNA has a higher melting point
- RNA-DNA is intermediate to DNA-DNA and RNA-RNA
How do we take advantage of denaturation/ renaturation?
- Nucleic acid hybridization is the formation of a stable duplex between two complementary strands of nucleic acid utilizing hydrogen bonding between base pairs.
- Hybridization requires a specific hybridization temperature (Thyb) which is 25C lower than Tm generally
- Nucleic acids from different species can form hybrids
- The closer the species, the more hybridization there will be
- Human DNA hybridizes more with mouse DNA than with yeast or bacterial DNA
T-hyb
Renaturing/annealing(Ta)
making a stable duplex between two complementary strands this form can be achieved by taking Tm and going 25 degrees Celsius below to achieve annealing
How do we detect specific DNA or RNA sequences in the model of the presence of many other sequences?
- Using hybridization with a probe
What is a probe?
- A probe is an oligonucleotide (a probe and primer/s categories
of oligos) - A probe has a specific sequence that is complementary to a specific stretch of DNA or RNA
- A probe must carry a fluorescent tag or a radioactive tag so that it can be detected
- The sequence of interest can be obtained from databases such as those found in NCBI
Describe Gel electrophoresis and how it relates to DNA.
-Agarose( derived from kelp , like jello does not disrupt base pairing
-agarose gel is placed in an electric file d
-DNA is negatively charged and travels toward the positive electrode( the backbone due to phosphate)
-Larger molecules move more slowly than bigger ones
-separate nucleic acids by size
-include a marker( set of of bands of different known sizes)
Describe how a Nucleic acid sample is taken and detected with a mixture of other DNA or RNA.
- Mixture of diffrent DNAs or RNAS
- Run on an agarose gel using electrophoresis
- Transfer the DNA to nitrocellulose membrane
- hybridize with labeled probe ( Temp of hybd)
- Use an autoradiogram to identify what the probe picked up or a fluorescent instrument
- When trying to detect DNA (southern blot) and RNA (northern blot)
What is southern blotting?
-detect DNA
-identify crime scene individual ( hair samples)
-predicting the onset of disease
What is northern blotting?
identify mRNA expression in different tissues(expression happens only on certain cells)
-is important to establish baseline controls (to distinguish negative results from bad experiments)
-sequencing can answer the same question but these techniques are still very often used
How do you calculate Tm?
Basic melting temperature Tm calculations
-sequences less than 14 nucleotides
(A+T)2 + (G+C)4
-sequences longer than 13 nucleotides
Tm=64.9+41*(G+C-16.4)/(A+T+G+C)
How do you calculate Thyb when you have Tm?
Thyb=Tm-25
Nucleotides and nucleic acids can undergo which type of transformation?
uncatalyzed chemical transformations
Can nucleotides undergo spontaneous alternations in covalent structures?
yes,
the rate of these is slow but relevant because cells have a low tolerance for genetic changes.
What is an alteration in the DNA structure that produces permanent genetic change?
Mutations
process of aging and carcinogenesis is linked to the accumulation of mutations.
Deamination
*Is a natural process
*sees the spontanoues loss of exocyclic amiono groups
*very common with Cytosine(DNA)–> Uracil
-1 in 107 Cs in 24 hours per cell
describe cytosine deamination
*repair mechanism in cells can readily recognize uracil and remove it
* If DNA contained uracil normal rather than thymine deamination of cytosine would be more problematic
*APOBEC’s
APOBEC’s
A family of proteins found in human cells that deaminate viral genomes such as HIV rendered it nonviable
-HIV has evolved a protein called Vif which binds to APOBEC and triggers degradation of this protein thus fighting the deamination
-Vif is an important target for antiviral medicine
When is DNA enzymatically methylated?
usually after DNA synthesis
Where does methylation tend to occur
As and Cs more than Gs
Where is methylation confined to
hotspots like CpG sequences
What is the function of methylation?
-methylation tends to block gene expression (transcription of DNA to RNA)
-Hypermethylation can be associated with cancer with silence genes normally controlling cell growth
How is methylation catalyzed?
enzyme called methylases do the job and they need S-adenosylmethionine as a methyl group donor
What does methylation do in bacteria?
Methtlylaiton signals ‘self” DNA ( remember restriction enzymes)
How can polymer sequences of DNA and RNA be synthesized with automated procedures involving chemical and enzymatic methods?
Solid-phase synthesis of DNA and RNA occurs in the
3’—-5’ direction
What forms of DNA and RNA can be synthesized?
ssDNA (primers are cheap and easy to manufacture)
dsDNA
ssRNA
What is a genome?
The complement of genetic information in a cell- one complete copy of the information required to specify that organism
when looking at a genome what does a molecular biologist care about most?
the function of or a few genes in the genome
About how much DNA is protein-coding?
1.5% or -21,000 genes
*if you include introns in the count about 30% of the genome contains genes (introns are components that exist in the pre-mRNA step)
How many protein-coding genes in chromosome 1?
2,000-2,100
how many base pairs are in the human genome?
3 billion base pairs
What is the average size of the protein-coding gene in the human genome?
20,000 nt
What year did pioneering techniques for MCB start?
1970
What were some of the pioneered techniques for MCB?
-Techniques for DNA cloning paved the way for the modern field of genomics, transcriptomic, and proteomics
-the study of genes, mRNA transcripts, and proteins on the scale of whole cells and organisms
ex.PCR how we test for the presence of COVID
Recombinant DNA
DNA that has been formed artificially by combining constituents from different organisms
What is the objective of Recombinant DNA?
-Isolate genes for study
-identify new genes and proteins
-characterize genes
-modify genes: correct defects in DNA
-Re-expressing gens in other hosts or organisms
ex. manufacturing of large quantities of specific gene products such as hormones, vaccines, and other biological agents of medical interest.
What is cloning?
-A clone is an identical copy
-the term was originally applied to cells produced when a cell of a single type was isolated and allowed to reproduce to create a population of identical cells
Describe the 5 steps for cloning.
- obtain the DNA segment to be cloned: use restriction endonuclease as molecular scissors
- selecting a small molecule of DNA capable of self-replication: cloning vectors/carrier
- joining two DNA fragments using DNA ligase - recombinant DNA
- moving recombinant DNA from the test tube to a host: use bacteria: Transformation
- selecting or identifying host cells that contain the recombinant DNA: antibiotic resistance
What is the molar extinction coefficient
measures how strongly a substance absorbs light at a particular wavelength
Beers law -> A=Ecl
Are covalent bonds affected during the denaturation of double-helical DNA and RNA?
no the phosphodiester bonds remain intact
How can bacterial cells be made competent for a transformation mechanism?
By treatment with cold CaCl followed by a brief heat shock at 37-43 degrees Celsius. ( this mechanism isn’t well understood but this allows for the take up of plasmid)
Endonucleases
recognize DNA at specific recognition sequences( or restriction sites)
cleave it to generate a set of smaller fragments
What is the opposite of endonuclease?
DNA ligase
-DNA fragment of interest can be joined to the DNA of a suitable cloning vector
Type 2 restriction endonuclease
cleaves DNA at specific base sequences
DNA ligase
Joins two DNA molecules or fragments
What is being cut when DNA is broken off?
The backbone
Name the two forms of using ligation
Sticky end ligation and Blunt end ligation
If you cut DNA can it be put back?
Yes its a process we call ligase
What does sticky end ligation look like?
5’ G |AATT C 3’
‘’’’’’’’’’’’’
3’ C TTAA |G 5’
it cleaves off and has loose ends that are single-stranded
but this type of ligation is more efficient than blunt end ligation
What does blunt end cleavage look like?
It cuts down right in the middle breaking off with no loose strands with the formation of a Phosphate and OH group on each of the ends
How do researchers create new DNA sequences?
By inserting synthetic DNA fragments called linkers, between the ends that are being ligated
What is a polylinker?
Inserted DNA fragments with multiple recognition sequences for restriction endonucleases( often DNA by cleavage useful later in the experiment as a point for inserting additional and ligation)
What is the model used to engineer polylinkers?
Plasmids
What is a cloning vector?
It is a DNA molecule that has an origin of replication and is capable of replicating and additional features that are important is antibiotic resistance
What is the function of Cloning vectors?
Allow amplification of inserted DNA segments
Name the types of vectors.
-Genetically engineered plasmid or phages
-bacterial artificial chromosomes (BAC)
-yeast artificial chromosomes ( YAC)
Plasmid
-Small circular, extrachromosomal DNA segment: replicates independently of the chromosome
Where is plasmid found?
bacteria in a wider variety (5000-40000bp)
The number of genes between plasmids can vary from one, few, or many ( true or false)
true
What mechanism are plasmids capable of generating in bacteria?
-Antibiotics resistance
-allowing some bacteria ability to colonize plants
What are some of the tools Molecular biologists have developed through the use of plasmids?
-Allow controlled expression of genes
-allowing cloning of genes
Can nucleotides above 200 be synthesized?
it is inefficient and not doable beyond 200
-we can make short oligonucleotides easily in vitro
Why is sticky end ligation important?
It allows for the directionality of cloning
-sometimes you need to express a protein and you have to have ATG start left to right
Where do lab-modified plasmids stem from?
All modified plasmids stem from an existing plasmid
What is the natural process from bacteria that we have stolen to develop cloning technology?
Transformation
What is the process called when small plasmids can be introduced into bacterial cells?
Transformation
What cell is often used for transformation in the lab?
E. Coli ( but other bacteria are also used)
What is an alternative method for getting plasmids into a cell?
Cells incubated with plasmid DNA are subjected to a high voltage pulse. this is called electroporation allows for the membrane to become permeable to large molecules
What forms can plasmid exist in?
Supercoiled and Relaxed circle( pickled)
Why is important to cut the plasmid before running a gel?
Plasmid can exist in a combination of forms and by not cutting it the traveling bands aren’t an accurate representation of how large the DNA is.
Ori
Orging of replication: required to propagate the plasmid
What happens if you cut a portion of Plasmid that expresses antibiotic resistance?
This will lead to the loss of resistance the issue happens when restriction-cutting sites are present in segments that express antibiotic resistance.
How can you facilitate a better entry into a cell with a plasmid?
making the plasmid smaller so by trimming away DNA segments from a larger parent plasmid getting rid of portions that the researcher may not need.
What does ampicillin(Amp, Ap) do?
Inhibits cell wall formation: inactivated by beta-lactamase
What do other antibiotics like Hygromycin neomycin and tetracycline do?
They inhibit translation in bacteria which is known to block the mechanism that allows for protein creation
How big can an E. coli plasmid vector clone be?
up to 10kb
What cloning vector would be better for large sizes of cloning?
BAC
-often used in sequencing
-can be stably maintained in bacteria
What is an Insert
What you are trying to clone, a piece of DNA from any gene, you insert it into a vector. It is cut out of a chromosome using restriction enzymes
Amplicon
a piece of DNA amplified by PCR
Template
that which you are amplifying (PCR template- DNA that you are amplifying by PCR)
Why isn’t it practical to use restriction enzymes to cut out the insert?
Introduce the restriction site that you want using PCR
PCR
Polymerase Chain reaction
What is PCR amplification?
DNA replication in a test tube
what is needed to amplify the region of DNA?
Forward primer and reverse primer
Explain the process that occurs for PCR amplification.
- Heat is applied to separate the strands
- the addition of synthetic oligonucleotide primers: cool.
- Add a thermostable DNA polymerase to catalyze 5’->3’ DNA synthesis
- repeat steps 1 and 2
Why is important to know the sequence of the target?
this allows you to design and synthesize primers
DNA polymerase requires?
-an already existing nucleotide chain to bind and add nucleotides one at a time
-add nucleotides building blocks like dNTPs
-special buffer to maintain pH, salts, and MgCl2
What are the 3 principles PCR relies on from MCB?
Denaturation: melting double-stranded DNA template in single strands:95 degrees Celsius
Annealing: complementary DNA strand hybridization via DNA primers:(Ta=Tm-5c)
—complementary primer to the 3’ ends of the target
—lower temperature and primer at very high concentration
extension: DNA strand synthesis via DNA polymerase and dNTPs;68-72 C
Each PCR cycle includes the above 3 steps
after 20 cycles the DNA segment has been amplified 2^20 or million-fold if the reaction conditions are ideal
How many copies can come from a cycle that runs 36 times?
2^36= 68 billion copies
Thermus aquaticus ( TAQ) polymerase
this is the enzyme that does not denature at high temperatures and allows for polymerase to be possible
does not denature at 95 C
What are the utilities for PCR?
-this tech is highly sensitive: PCR can detect and amplify as little as one DNA molecule in almost any type of sample including ancient ones
-PCR has allowed for the cloning of samples from 40,000 years old and is used to clone DNA from mummified remains of humans and extinct animals. ex. woolly mammoth
-been used for detecting viral infections before the cause of symptoms
-prenatal diagnosis of a wide array of genetic disease
How can you quantify DNA when doing PCR protocols to estimate relative copies?
qPCR
How is the amount determined in a qPCR product?
-by measuring the level of a fluorescent probe attached to a reporter oligonucleotide complementary to the DNA segment that is being amplified
-using a fluorescent dye that incorporates into newly made DNA
Is standard PCR quantitative?
no
How do you determine what sample is most abundant in qPCR?
the most abundant is the first to cross the CT
What is CT?
the cycle number at which the threshold is first surpassed
No template in qPCR is significant because?
the line follows the slow increase in background signal observed in a control that does not include an added sample of DNA. seeing this in the COVID test means the sample isn’t replicated meaning it is negative.
How is RNA made viable for qPCR?
Reverse Transcription(RT) using reverse transcriptase and a primer
Why are we able to RT RNA?
Most RNA have a poly-A tail which allows the formation of a brief period of A form mRNA-DNA hybrid which can then become DNA
The Sanger method
identifies nucleotide sequences in cloned genes
sanger’s method is also known as?
dideoxy chain-termination method
Describe the Sanger method
-This method makes use of the mechanism of DNA synthesis by DNA polymerases
-it requires the enzymatic synthesis of a DNA strand complementary to the strand under analysis using a radioactively labeled primer
-the 3-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond
-nucleotide analogs called dideoxynucleotide triphosphate (ddNTPs)interrupt DNA synthesis because of their lack of the 3’ hydroxyl group needed for the next step
Before the 1970s it was laborious to determine the size of nucleic acid. What was the size they were able to determine?
5 or 10 nucleotides
Who were the scientists who developed the two techniques in 1977 to better sequence larger DNA molecules?
-Allan Maxam and Walter Gilbert
-Frederick Sanger
What material is used instead of agarose when working with DNA oligonucleotides(up to a few hundred nucleotides)?
polyacrylamide because it enables researchers to detect small size differences between DNA fragments.
Describe DNA synthesis via the dNTP point of view.
involves a reaction between the 3’hydroxly group of the primer dNTP and the phosphate group of an incoming dNTP
In a sequencing reaction, what is contained to carry out DNA synthesis?
a mixture of dNTPs and ddNTP-radiolabeled primer
different ddNTP is used in each reaction.
How is nucleotide sequenced determined after sequencing?
analyzed by autoradiography
How was the Sanger methods automated?
A variation of the Sanger method in which each of the four dideoxynucleotides used for a reaction was labeled with a differently colored fluorescent tag
With automation how fast can researched sequences in the human genome?
In a few hours, DNA molecules contain thousands of nucleotides
ex. researcher sequence all 3.2X109 bp of DNA In a human cell.
How is the information acquired via the Sanger method?
-each ddNTP is linked to a fluorescent dye that gives the same color to all the fragment’s termination ends. Each nucleotide has its color
-all four labeled ddNTPs are added together
-the colored fragments are separated by size in a gel capillary tube which allows for faster separation
-All fragments travel based on their given length and the color is detected and recorded to interpret the sequence of DNA
What is next-gen sequencing capable of?
-complete genome in a day or two, bacterial in a few hours
-personal genomic sequence leads to personalized medicine
-modified miniaturization of the Sanger method to upscale the procedure
Expression vectors
cloning vector with the transcription and translation signals needed for the regulated expression of a cloned gene
what vectors allow for transcription?
Bacterial promoter(P) and operator (O) sequences
what vectors allow for translation?
ribosomes binding sites
Where are well-characterized promoters and regulatory elements positioned?
Near several unique restriction sites for cloning, this is so genes inserted at restriction sites will be expressed from the regulated promoter elements.
What organism can be used to express recombinant proteins?
Any organism from different heterologous species
-Bacteria especially are highly understood, easy to grow, and cheap
-yeast
-insects
-mammalian cell in culture
-transgenic animals
How are you able to retrieve a specific protein in a mixture of many proteins from your model organism?
Terminal Tags provide handles for affinity purification.
