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S25 Molecular Biology > L4 Techniques of Molecular Biology: Analysis of DNA and RNA > Flashcards

L4 Techniques of Molecular Biology: Analysis of DNA and RNA Flashcards

1
Q

what do you use for the physical separation of nucleic acids?

A

gel electrophoresis

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2
Q

gel electrophoresis - how does it work

A
  1. run it through agarose (porous, gel matrix)
  2. negative nucleic acids run to the positive pole
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3
Q

gel electrophoresis - how to visualize cells

A

Ethidium bromide:
- a fluorescent dye that binds to DNA (via intercalation)
- visible under UV light

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4
Q

gel electrophoresis: ethidium bromide - what is intercalation

A

the DNA soaks up the ethidium bromide like a sponge

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5
Q

gel electrophoresis - what does it separate DNA by

A
  • size (number of base pairs)
  • toplogy
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6
Q

gel electrophoresis - DNA separation by size

A
  • need to add a molecular ladder in to show the sizes of DNA for comparison
  • how it works: the agarose has pores and the smaller ones go in faster and land towards the bottom
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7
Q

gel electrophoresis - DNA separation by topology

A
  • from high (big) to low (small):
    1. nicked/relaxed circle
    2. linear
    3. supercoiled
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8
Q

how can you cut DNA in vitro?

A

restriction enzymes

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9
Q

how can you cut DNA in vitro? - what is a restriction enzyme?

A
  • they cleave DNA at specific sequences (recognition sites)
  • after cleaving, they make “sticky ends” and a staggered cut
  • can be used to make recombinant DNA
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10
Q

restrictive enzymes - what are “sticky ends”

A
  • the cut DNA ends that matches the nucleotide sequences through base pairing
  • the two sticky ends come together manually
  • but need to insert ligase to make seal the ends bc there are no phosphodiester linkages
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11
Q

restrictive enzymes - what does to have a staggered cut

A

it cuts DNA in a way that leaves short, single-stranded overhangs on the resulting DNA fragments

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12
Q

example of a restriction enzyme - EcoRI (E. coli Restrictive Enzyme I)

A
  • it cuts in a palindrome manner (5’-3’ says the same ‘word’ as the other strand in 5’-3’)
  • sticky ends are sealed or can interact with another DNA of the same enzyme to make recombinant DNA
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13
Q

how can you produce many copies of a DNA fragment?

A

DNA cloning

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14
Q

DNA cloning - how is DNA amplified?

A
  • DNA is usually amplified/cloned by two main techniques:
    1. polymerase chain reaction (PCR)
    2. inserted into a vector for in vivo propagation (microorganism)
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15
Q

DNA cloning - what is a vector

A
  • used to make copies of a foreign piece of DNA
  • often is a circular piece of DNA called a plasmid
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16
Q

DNA cloning - traits of a vector

A
  • origin of replication (needed so it can be propagated in a microorganism)
  • selectable marker (to detect/track the presence of the vector)
  • unique restriction enzyme recognition sites (to introduce foreign DNA)
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17
Q

DNA cloning: traits of a vector - how do you choose the selectable marker?

A
  • need to pick one that will establish conditions where the plasmid will only grow if the bacteria has the plasmid
  • the conditions need to select for the plasmid
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18
Q

DNA cloning - vector example

A
  • pBluescript
  • has:
    1. an E. coli origin of replication
    2. a marker (ampicillin resistance gene - will select for plasmid growth)
    3. restrictive enzyme sites via multiple cloning sites
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19
Q

DNA cloning - how do you make a vector?

A
  1. cut the plasmid via restrictive enzyme
  2. sticky ends have compatibility with foreign DNA –> becomes a recombinant plasmid
  3. use the selective marker so the plasmid grows due to selection
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20
Q

how can you identify a specific DNA molecule?

A

hybridization

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21
Q

hybridization

A
  • base-pairing between complementary single-stranded nucleic acids
  • need to design a probe
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22
Q

hybridization - what is a probe

A
  • a purified or synthesized DNA molecule used to search mixtures of nucleic acids for complementary molecules
  • must be labeled by fluorescence or radioactivity
  • can be used to identify DNA/RNA separated by electrophoresis
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23
Q

what are the types of hybridization?

A
  • southern blot hybridization (DNA)
  • northern blot hybridization (RNA)
  • microarray analysis (RNA)
  • RNA in situ hybridization (RNA)
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24
Q

types of hybridization - southern blot hybridization

A
  • looks at DNA
  • asks: is a foreign piece of DNA inserted into a genome of interests
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25
Q

types of hybridization - how to do a southern blot hybridization

A
  1. cut the genome with a restriction enzyme
  2. separate by electrophoresis
  3. transfer or “blot” to a membrane (to allow physical manipulation)
  4. apply probe
  5. detect probe (autoradiogram, etc.)
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26
Q

types of hybridization: southern blot hybridization - what will the PCR results look like without the probe?

A
  • smeared
  • this is bc the restriction enzyme is randomly chewing up the DNA
27
Q

types of hybridization - northern blot hybridization

A
  • looks at RNA
  • asks: when, where, and/or how strongly is a given gene expressed
  • similar to southern blot but RNA is not cut since it already exists in manageable sizes
  • can still use a labeled DNA probe due to RNA-DNA hybridization
  • needs a “loading control”
28
Q

types of hybridization: northern blot hybridization - what is a loading control

A
  • to see the difference in expression
  • if there is no loading control, you cannot make inferences/conclusions
29
Q

types of hybridization: northern blot hybridization - how are the results read?

A

intensity of band = level of gene expression

30
Q

types of hybridization - microarray analysis

A
  • looks at RNA
  • asks: when, where, and/or how strongly is every gene in the genome expressed?
  • utilizes reverse hybridization
31
Q

types of hybridization: microarray analysis - what is reverse hybridization

A

attaching known DNA sequences in a spotted array which represents every gene in the genome

32
Q

types of hybridization: microarray analysis - how is it done

A
  1. reverse hybridization: one probe for every gene within the genome
  2. isolate mRNA
  3. make complementary DNA (cDNA) via reverse transcriptase
  4. label and apply to a slide
  5. measure the intensity of cDNA hybridized to each spot
  6. readout of expression levels for all genes
33
Q

types of hybridization: microarray analysis steps - selectively isolate mRNA

A

need to selectively isolate mRNA with poly A tails

34
Q

types of hybridization: microarray analysis steps - make cDNA via reverse transcriptase

A
  • to do this, take the isolated mRNA w poly A tail
  • have it base pair with a poly T sequence
35
Q

types of hybridization: microarray analysis steps - why do we use cDNA instead of mRNA

A

mRNA is not easy to work with

36
Q

types of hybridization: microarray analysis steps - label cDNA and apply to array

A
  • can label the cDNA with fluorescence
  • take the two sample groups and use reverse transcriptase labeling
  • next combine the targets
  • finally, hybridize to the microarray
37
Q

types of hybridization: microarray analysis - what does the results look like

A
  • a cluster analysis of data (heat map)
  • the color can represent 4 things:
    1. red: a gene expressed higher in sample 1
    2. green: a gene expressed higher in sample 2
    3. yellow: a gene expressed equally in sample 1 and 2
    4. dark shade: no expression
38
Q

types of hybridization - RNA in situ hybridization

A
  • looks at RNA
  • asks: in precisely what tissue types is a given gene expressed?
  • shows where the gene is expressed at the cellular level
39
Q

types of hybridization: RNA in situ hybridization - how is it done

A
  1. tissue is chemically fixed and section into thin slices
  2. endogenous RNA is fixed in place (in situ) and exposed
  3. a labeled RNA probe (for a specific gene of interest) is applied and hybridizes to complementary mRNA
  4. the label then allows visualization of probe via an enzymatic reaction
40
Q

how can you amplify a specific DNA molecule in vitro?

A

polymerase chain reaction (PCR)

41
Q

what is PCR?

A
  • an in vitro reaction that uses DNA polymerase to replicate DNA molecules
  • it generates many identical copies of a particular DNA
  • is a method for DNA cloning
42
Q

PCR steps

A
  1. denaturation
  2. primer annealing
  3. extension
43
Q

PCR steps - denaturation

A
  • strands of the DNA template are denatured by heat at 95 degrees C
44
Q

PCR steps - primer annealing

A
  • cool to 50 degrees C - 60 degrees C to allow primer to bind to region
  • primers then bind/anneal to complementary region
  • via adding a 3’ hydroxyl group to DNA to allow DNA polymerase to replicate the DNA
  • also shows which part of the DNA will be amplified
45
Q

PCR steps - extension

A
  • reaction is heated to 70 degrees C
  • the DNA polymerase will extend primers to synthesize the DNA
46
Q

PCR steps - what is the result?

A
  • the steps are then repeated to yield the required number of DNA copies
  • each step results in DNA number doubling
47
Q

How can you use PCR to assess RNA expression levels?

A
  • Reverse Transcription Polymerase Chain Reaction (RT-PCR)
  • Quantitative real-time polymerase chain reaction (qPCR or qRT-PCR)
48
Q

Reverse Transcription Polymerase Chain Reaction (RT-PCR) - how does it work

A
  • cDNA is used as the template
  • amount of product amplified correlates with starting template
  • reaction can be:
    1. Semi-quantitative
    2. Quantitative
49
Q

Reverse Transcription Polymerase Chain Reaction (RT-PCR) - what is the difference between semi-quantitative and quantitative

A
  • semi-quantitative: which one has more expression or not
  • quantitative: quantifying the specific expression level (qRT-PCR)
50
Q

Quantitative real-time polymerase chain reaction (qPCR or qRT-PCR)

A
  • PCR where a targeted DNA molecule is amplified and
    simultaneously detected
  • allows quantification of starting amount of target DNA
  • can be used to assess gene expression levels (starting amount = expression level)
  • RNA is isolated, converted to cDNA and use as template in qPCR
51
Q

Quantitative real-time polymerase chain reaction (qPCR or qRT-PCR) - how does it work?

A
  • Use the fluorescent dye (SYBR green) for detection of dsDNA product
  • As double stranded PCR are generated, dye is incorporated into it and can you detect DNA
  • method quantifies it in real time via a amplification plot
52
Q

Quantitative real-time polymerase chain reaction (qPCR or qRT-PCR) - explain the amplification plot

A
  • two phases: (1) exponential and (2) plateau phase
  • has a threshold cycle (Ct)
53
Q

Quantitative real-time polymerase chain reaction (qPCR or qRT-PCR) - what is the threshold cycle

A
  • number at which the amplification plot crosses a threshold level of fluorescence
  • Ct is inversely proportional to starting amount of template (lower Ct –> higher starting amount)
54
Q

How do you determine the sequence of a DNA fragment?

A

Sanger Dideoxy DNA Sequencing

55
Q

Sanger Dideoxy DNA Sequencing

A
  • an in vitro DNA synthesis reaction
  • contains both deoxyribonucleoside triphosphates (dNTPs) and dideoxyribonucleoside triphosphates (ddNTPs)
56
Q

Sanger Dideoxy DNA Sequencing - whats the difference between dNTPs and ddNTPs?

A
  • dNTPs lack 2’ OH
  • ddNTPs lack both 2’ OH and 3’ OH
57
Q

Sanger Dideoxy DNA Sequencing - how does it work

A
  1. incubate reaction mixture
  2. DNA synthesis occurs
  3. collect DNA strands
  4. seperate fragments
  5. read output
58
Q

Sanger Dideoxy DNA Sequencing - incubate reaction mixture

A

mixture of:
- dNTPs to extend the DNA strand
- ddNTPs to terminate synthesis

59
Q

Sanger Dideoxy DNA Sequencing - DNA synthesis occurs

A

each strand is unique with varying length since ddNTPs are added in at different times

60
Q

How do you determine the sequence of a genome?

A
  • Shotgun Sequencing
  • Next Generation Sequencing
61
Q

Shotgun Sequencing

A
  • Genome is sheared into random fragments, cloned into a vector and sequenced individually
  • Sequences are then assembled computationally
62
Q

Next Generation Sequencing

A
  • high throughput sequencing using modern technologies
    ex:
  • Illumina (Solexa)
  • Transcriptome
63
Q

Next Generation Sequencing - Illumina (Solexa) Sequencing

A
  • simultaneous sequencing of millions of DNA templates adhered to solid surface
  • add DNA pol and fluorescently label the dNTPs (with terminator so only one base added at a time)
  • added base is detected, terminator removed, process is repeated
64
Q

Next Generation Sequencing - Transcriptome (RNA-seq)

A
  • sequence cDNA sample with Next Generation Sequencing
  • “map” data onto a reference genome
  • shows expression levels

S25 Molecular Biology (7 decks)

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