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Exam 2 Flashcards

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1
Q

Explain DNA as an information carrier.

A

-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

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2
Q

What grooves does DNA form and why do they matter?

A

Major and minor
-these are sites of interactions with protein and nucleic acids

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3
Q

What is the most common helix form of DNA?

A

Right-handed helix

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4
Q

what is the directionality of the DNA double helix?

A

Antiparallel strands: Opposite directionality

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5
Q

How many base pairs make up the most common Helix?

A

Most common B has a range of 10.1-10.5 bp/turn
roughly 10

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6
Q

What are other forms of DNA?
Describe the direction, occurrence, and condition

A

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

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7
Q

What is a palindrome?

A

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’

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8
Q

What is an inverted repeat?

A

-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

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9
Q

What is a mirror repeat?

A

-Inverted repeat sequence is nonpalindromic

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10
Q

What is the biological significance of different DNA structures?

A

-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

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11
Q

primary structure different from DNA and RNA

A

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

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12
Q

When and how was the structure of tRNA discovered?

A

in the 1970s with the use of X-ray crystallography

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13
Q

RNA is generally single stranded name the an exception

A

in dsRNA genomes of some virus

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14
Q

RNA does not always adopt a specific 3-D structure

A

True

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15
Q

What can happen to the covalent back bond of DNA and RNA naturally?

A

slow nonenzymatic hydrolysis of the phosphodiester bonds

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16
Q

Who is most readily hydrolyzed under alkaline(basic) conditions in a test tube

A

RNA

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17
Q

What happens when a single-stranded RNA folds upon itself?

A

-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

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18
Q

What are examples of NON-watson crick base pairing in RNA?

A

Two Adenine coming together
Guanine and Uracil getting together

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19
Q

How are we able to measure DNA?

A

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

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20
Q

How do you break the phosphodiester bond backbone?(DNA or RNA)

A
  • 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
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21
Q

How do you break the duplex of DNA or RNA?

A

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.

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22
Q

Describe the denaturation of double-helical DNA and RNA

A

-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

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23
Q

Tm

A

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

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23
Q

Explain the relationship between DNA and Tm

A

*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

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23
Q

Describe the interaction of DNA with UV light when experiencing changes

A
  • 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
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24
Q

What is renaturation?

A

*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

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25
Q

What is an extension?

A

The copying of DNA

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26
Q

In controlled conditions which base of complimentary bases will denature first?

A

AT

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27
Q

Why is strand separation crucial?

A

DNA replication, Transcription

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28
Q

Describe the trends of melting points seen in DNA and RNA

A
  • 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
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29
Q

How do we take advantage of denaturation/ renaturation?

A
  • 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
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30
Q

T-hyb

A

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

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31
Q

How do we detect specific DNA or RNA sequences in the model of the presence of many other sequences?

A
  • Using hybridization with a probe
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32
Q

What is a probe?

A
  • 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
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33
Q

Describe Gel electrophoresis and how it relates to DNA.

A

-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)

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34
Q

Describe how a Nucleic acid sample is taken and detected with a mixture of other DNA or RNA.

A
  1. Mixture of diffrent DNAs or RNAS
  2. Run on an agarose gel using electrophoresis
  3. Transfer the DNA to nitrocellulose membrane
  4. hybridize with labeled probe ( Temp of hybd)
  5. Use an autoradiogram to identify what the probe picked up or a fluorescent instrument
  6. When trying to detect DNA (southern blot) and RNA (northern blot)
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35
Q

What is southern blotting?

A

-detect DNA
-identify crime scene individual ( hair samples)
-predicting the onset of disease

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36
Q

What is northern blotting?

A

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

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37
Q

How do you calculate Tm?

A

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)

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38
Q

How do you calculate Thyb when you have Tm?

A

Thyb=Tm-25

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39
Q

Nucleotides and nucleic acids can undergo which type of transformation?

A

uncatalyzed chemical transformations

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40
Q

Can nucleotides undergo spontaneous alternations in covalent structures?

A

yes,
the rate of these is slow but relevant because cells have a low tolerance for genetic changes.

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41
Q

What is an alteration in the DNA structure that produces permanent genetic change?

A

Mutations
process of aging and carcinogenesis is linked to the accumulation of mutations.

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42
Q

Deamination

A

*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

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43
Q

describe cytosine deamination

A

*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

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44
Q

APOBEC’s

A

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

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45
Q

When is DNA enzymatically methylated?

A

usually after DNA synthesis

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46
Q

Where does methylation tend to occur

A

As and Cs more than Gs

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47
Q

Where is methylation confined to

A

hotspots like CpG sequences

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48
Q

What is the function of methylation?

A

-methylation tends to block gene expression (transcription of DNA to RNA)
-Hypermethylation can be associated with cancer with silence genes normally controlling cell growth

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49
Q

How is methylation catalyzed?

A

enzyme called methylases do the job and they need S-adenosylmethionine as a methyl group donor

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50
Q

What does methylation do in bacteria?

A

Methtlylaiton signals ‘self” DNA ( remember restriction enzymes)

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51
Q

How can polymer sequences of DNA and RNA be synthesized with automated procedures involving chemical and enzymatic methods?

A

Solid-phase synthesis of DNA and RNA occurs in the
3’—-5’ direction

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52
Q

What forms of DNA and RNA can be synthesized?

A

ssDNA (primers are cheap and easy to manufacture)
dsDNA
ssRNA

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53
Q

What is a genome?

A

The complement of genetic information in a cell- one complete copy of the information required to specify that organism

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54
Q

when looking at a genome what does a molecular biologist care about most?

A

the function of or a few genes in the genome

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55
Q

About how much DNA is protein-coding?

A

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)

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56
Q

How many protein-coding genes in chromosome 1?

A

2,000-2,100

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57
Q

how many base pairs are in the human genome?

A

3 billion base pairs

58
Q

What is the average size of the protein-coding gene in the human genome?

A

20,000 nt

59
Q

What year did pioneering techniques for MCB start?

A

1970

60
Q

What were some of the pioneered techniques for MCB?

A

-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

61
Q

Recombinant DNA

A

DNA that has been formed artificially by combining constituents from different organisms

62
Q

What is the objective of Recombinant DNA?

A

-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.

63
Q

What is cloning?

A

-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

64
Q

Describe the 5 steps for cloning.

A
  1. obtain the DNA segment to be cloned: use restriction endonuclease as molecular scissors
  2. selecting a small molecule of DNA capable of self-replication: cloning vectors/carrier
  3. joining two DNA fragments using DNA ligase - recombinant DNA
  4. moving recombinant DNA from the test tube to a host: use bacteria: Transformation
  5. selecting or identifying host cells that contain the recombinant DNA: antibiotic resistance
65
Q

What is the molar extinction coefficient

A

measures how strongly a substance absorbs light at a particular wavelength
Beers law -> A=Ecl

66
Q

Are covalent bonds affected during the denaturation of double-helical DNA and RNA?

A

no the phosphodiester bonds remain intact

67
Q

How can bacterial cells be made competent for a transformation mechanism?

A

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)

68
Q

Endonucleases

A

recognize DNA at specific recognition sequences( or restriction sites)
cleave it to generate a set of smaller fragments

69
Q

What is the opposite of endonuclease?

A

DNA ligase
-DNA fragment of interest can be joined to the DNA of a suitable cloning vector

70
Q

Type 2 restriction endonuclease

A

cleaves DNA at specific base sequences

71
Q

DNA ligase

A

Joins two DNA molecules or fragments

72
Q

What is being cut when DNA is broken off?

A

The backbone

73
Q

Name the two forms of using ligation

A

Sticky end ligation and Blunt end ligation

74
Q

If you cut DNA can it be put back?

A

Yes its a process we call ligase

75
Q

What does sticky end ligation look like?

A

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

76
Q

What does blunt end cleavage look like?

A

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

77
Q

How do researchers create new DNA sequences?

A

By inserting synthetic DNA fragments called linkers, between the ends that are being ligated

78
Q

What is a polylinker?

A

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)

79
Q

What is the model used to engineer polylinkers?

A

Plasmids

80
Q

What is a cloning vector?

A

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

81
Q

What is the function of Cloning vectors?

A

Allow amplification of inserted DNA segments

82
Q

Name the types of vectors.

A

-Genetically engineered plasmid or phages
-bacterial artificial chromosomes (BAC)
-yeast artificial chromosomes ( YAC)

83
Q

Plasmid

A

-Small circular, extrachromosomal DNA segment: replicates independently of the chromosome

84
Q

Where is plasmid found?

A

bacteria in a wider variety (5000-40000bp)

85
Q

The number of genes between plasmids can vary from one, few, or many ( true or false)

A

true

86
Q

What mechanism are plasmids capable of generating in bacteria?

A

-Antibiotics resistance
-allowing some bacteria ability to colonize plants

87
Q

What are some of the tools Molecular biologists have developed through the use of plasmids?

A

-Allow controlled expression of genes
-allowing cloning of genes

88
Q

Can nucleotides above 200 be synthesized?

A

it is inefficient and not doable beyond 200
-we can make short oligonucleotides easily in vitro

89
Q

Why is sticky end ligation important?

A

It allows for the directionality of cloning
-sometimes you need to express a protein and you have to have ATG start left to right

90
Q

Where do lab-modified plasmids stem from?

A

All modified plasmids stem from an existing plasmid

91
Q

What is the natural process from bacteria that we have stolen to develop cloning technology?

A

Transformation

92
Q

What is the process called when small plasmids can be introduced into bacterial cells?

A

Transformation

93
Q

What cell is often used for transformation in the lab?

A

E. Coli ( but other bacteria are also used)

94
Q

What is an alternative method for getting plasmids into a cell?

A

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

95
Q

What forms can plasmid exist in?

A

Supercoiled and Relaxed circle( pickled)

96
Q

Why is important to cut the plasmid before running a gel?

A

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.

97
Q

Ori

A

Orging of replication: required to propagate the plasmid

98
Q

What happens if you cut a portion of Plasmid that expresses antibiotic resistance?

A

This will lead to the loss of resistance the issue happens when restriction-cutting sites are present in segments that express antibiotic resistance.

99
Q

How can you facilitate a better entry into a cell with a plasmid?

A

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.

100
Q

What does ampicillin(Amp, Ap) do?

A

Inhibits cell wall formation: inactivated by beta-lactamase

101
Q

What do other antibiotics like Hygromycin neomycin and tetracycline do?

A

They inhibit translation in bacteria which is known to block the mechanism that allows for protein creation

102
Q

How big can an E. coli plasmid vector clone be?

A

up to 10kb

103
Q

What cloning vector would be better for large sizes of cloning?

A

BAC
-often used in sequencing
-can be stably maintained in bacteria

104
Q

What is an Insert

A

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

105
Q

Amplicon

A

a piece of DNA amplified by PCR

106
Q

Template

A

that which you are amplifying (PCR template- DNA that you are amplifying by PCR)

107
Q

Why isn’t it practical to use restriction enzymes to cut out the insert?

A

Introduce the restriction site that you want using PCR

108
Q

PCR

A

Polymerase Chain reaction

109
Q

What is PCR amplification?

A

DNA replication in a test tube

110
Q

what is needed to amplify the region of DNA?

A

Forward primer and reverse primer

111
Q

Explain the process that occurs for PCR amplification.

A
  1. Heat is applied to separate the strands
  2. the addition of synthetic oligonucleotide primers: cool.
  3. Add a thermostable DNA polymerase to catalyze 5’->3’ DNA synthesis
  4. repeat steps 1 and 2
112
Q

Why is important to know the sequence of the target?

A

this allows you to design and synthesize primers

113
Q

DNA polymerase requires?

A

-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

114
Q

What are the 3 principles PCR relies on from MCB?

A

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

115
Q

How many copies can come from a cycle that runs 36 times?

A

2^36= 68 billion copies

116
Q

Thermus aquaticus ( TAQ) polymerase

A

this is the enzyme that does not denature at high temperatures and allows for polymerase to be possible
does not denature at 95 C

117
Q

What are the utilities for PCR?

A

-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

118
Q

How can you quantify DNA when doing PCR protocols to estimate relative copies?

A

qPCR

119
Q

How is the amount determined in a qPCR product?

A

-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

120
Q

Is standard PCR quantitative?

A

no

121
Q

How do you determine what sample is most abundant in qPCR?

A

the most abundant is the first to cross the CT

122
Q

What is CT?

A

the cycle number at which the threshold is first surpassed

123
Q

No template in qPCR is significant because?

A

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.

124
Q

How is RNA made viable for qPCR?

A

Reverse Transcription(RT) using reverse transcriptase and a primer

125
Q

Why are we able to RT RNA?

A

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

126
Q

The Sanger method

A

identifies nucleotide sequences in cloned genes

127
Q

sanger’s method is also known as?

A

dideoxy chain-termination method

128
Q

Describe the Sanger method

A

-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

129
Q

Before the 1970s it was laborious to determine the size of nucleic acid. What was the size they were able to determine?

A

5 or 10 nucleotides

130
Q

Who were the scientists who developed the two techniques in 1977 to better sequence larger DNA molecules?

A

-Allan Maxam and Walter Gilbert
-Frederick Sanger

131
Q

What material is used instead of agarose when working with DNA oligonucleotides(up to a few hundred nucleotides)?

A

polyacrylamide because it enables researchers to detect small size differences between DNA fragments.

132
Q

Describe DNA synthesis via the dNTP point of view.

A

involves a reaction between the 3’hydroxly group of the primer dNTP and the phosphate group of an incoming dNTP

133
Q

In a sequencing reaction, what is contained to carry out DNA synthesis?

A

a mixture of dNTPs and ddNTP-radiolabeled primer
different ddNTP is used in each reaction.

134
Q

How is nucleotide sequenced determined after sequencing?

A

analyzed by autoradiography

135
Q

How was the Sanger methods automated?

A

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

136
Q

With automation how fast can researched sequences in the human genome?

A

In a few hours, DNA molecules contain thousands of nucleotides
ex. researcher sequence all 3.2X109 bp of DNA In a human cell.

137
Q

How is the information acquired via the Sanger method?

A

-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

138
Q

What is next-gen sequencing capable of?

A

-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

139
Q

Expression vectors

A

cloning vector with the transcription and translation signals needed for the regulated expression of a cloned gene

140
Q

what vectors allow for transcription?

A

Bacterial promoter(P) and operator (O) sequences

141
Q

what vectors allow for translation?

A

ribosomes binding sites

142
Q

Where are well-characterized promoters and regulatory elements positioned?

A

Near several unique restriction sites for cloning, this is so genes inserted at restriction sites will be expressed from the regulated promoter elements.

143
Q

What organism can be used to express recombinant proteins?

A

Any organism from different heterologous species
-Bacteria especially are highly understood, easy to grow, and cheap
-yeast
-insects
-mammalian cell in culture
-transgenic animals

144
Q

How are you able to retrieve a specific protein in a mixture of many proteins from your model organism?

A

Terminal Tags provide handles for affinity purification.

Molecular Biology (5 decks)

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