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MOL. BIO LEC > MIDTERM LEC 3 > Flashcards

MIDTERM LEC 3 Flashcards

1
Q

→ Set of techniques & methods used to study and understand the structure, interaction, & characteristics of nucleic acids & proteins in biological systems

A

NUCLEIC ACID/PROTEIN ANALYSIS

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2
Q

NUCLEIC ACID/PROTEIN ANALYSIS 3 MAIN METHODS

A
  1. Restriction enzyme mapping of DNA
  2. CRISPR enzyme systems
  3. Hybridization technology
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3
Q
  • Involves restriction enzyme such as endonucleases
  • Used to identify the position of restriction sites in DNA fragments in relation to each other.
    o Used for DNA fingerprinting, paternity testing
  • Developed using small circular bacterial plasmids.
A

Restriction enzyme mapping of DNA

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4
Q

commonly used endonuclease in restriction enzyme mapping of DNA

A

TYPE II ENDONUCLEASE

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5
Q

*Resulting DIFFERENCES IN SIZE OR NUMBER OF RESTRICTION FRAGMENTS
*Useful in epidemiologic studies.
*The basis of the first molecular-based human ID and mapping methods.
*Clinical analysis of structural changes in chromosomes associated with disease

A

Restriction Fragment Length Polymorphisms (RFLPs)

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6
Q

RESTRICTION MAPPING USES/APPLICATIONS

A

1.Gives an idea of genome sequence
2. Used to map genomes when sequence information is unknown.
3. Can be used to introduce primers for sequencing.
4. Used for ID and characterization of naturally-occurring plasmids and to engineer the construction of recombinant plasmids.

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7
Q
  • Found in archaea and bacteria
  • guides a common enzyme to specific sites determined by RNA components
  • Useful for manipulation of both DNA sequence and RNA expression.
A

CRISPR enzyme systems

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8
Q

CRISPR stands for

A

CLUSTERED REGULARLY INTERSPACED REPEATS

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9
Q

CRISPR enzyme systems TYPES:
Type I & II - target ______

A

dsDNA

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10
Q

CRISPR enzyme systems TYPES:
Type III - target ______

A

ssDNA & RNA

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11
Q
  • used to analyze the nucleic acid content of an unknown sample
A

Hybridization TECHNOLOGY

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12
Q
  • Formation of H bonds between 2 complementary strands of nucleic acid.
A

HYBRIDIZATION

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13
Q

IN HYBRIDIZATION TECHNIQUE:
- First strand is _________

A

UNLABELED

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14
Q

TARGET: DNA
PROBE: NUCLEIC ACID (NA)
PURPOSE: Gene structure

A

Southern blot

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15
Q

TARGET: RNA
PROBE: NA
PURPOSE: Transcript structure, processing, gene expression

A

Northern blot

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16
Q

TARGET: PROTEIN
PROBE: PROTEIN
PURPOSE: Protein processing, gene expression

A

WESTERN BLOT

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17
Q

TARGET: PROTEIN
PROBE: DNA
PURPOSE: DNA – binding proteins, gene regulation

A

Southwestern blot

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18
Q

TARGET: PROTEIN
PROBE: PROTEIN
PURPOSE: Modification of western blot using enzymatic detection (PathHunter); also, detection of specific agriculturally important proteins

A

Eastern blot

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19
Q

TARGET: LIPIDS
PROBE: NONE
PURPOSE: Transfer high performance liquid chromatography (HPLC)- separate lipids to play difluoride (PVDF) membrane for analysis by mass spectrometry

A

Far-eastern blot

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20
Q

Southern blot – procedure was first reported by

A

Edwin Southern

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21
Q
  • ss fragment of nucleic acid/protein with a DETECTABLE SIGNAL that specifically binds to complementary sequences/target protein.
  • PURPOSE: identify one or more sequences of interest with a large amount of nucleic acids.
  • OTHER USED: modified nucleic acid, such as peptide nucleic acids and locked nucleic acids.
A

PROBE

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22
Q

TYPES OF PROBES?
- SOUTHERN BLOTS
- Complementary to the target gene

A

Nucleic Acid (NA) Probes (DNA probes)

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23
Q

TYPES OF PROBES?
- NORTHERN BLOTS
- Complementary to the target sequence

A

Nucleic Acid (NA) Probes (RNA probes)

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24
Q

TYPES OF PROBES?
- WESTERN BLOTS
- Antibodies bind to the target protein

A

Protein Probes

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25
Q

In order for the probe to bind: target NA has to contain the _________

A

SEQUENCE OF INTEREST

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26
Q

Denaturation of dsDNA probes before use:
- Heating the probe (TEMP & TIME?) in hybridization solution

A

95°C, 10 – 15min

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27
Q

Denaturation of dsDNA probes before use:
o Treating with 50% formamide/2x SSC at a lower temp. for a shorter time (TEMP & TIME?)

A

75°C; 5-6 min

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28
Q

 EARLY METHODS: fragment of gene was cloned on bacterial plasmid — isolated by restriction enzyme digestion and gel purification — labeling and denaturation of fragment — applied to southern/northern blot.
 Isolation of sequence of interest from viral genomes.
 In vitro organic synthesis of predetermined sequence — only for short, oligometric probes.
 Synthesized using PCR

A

DNA PROBES

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29
Q

must generate a detectable signal for visualization of the probe binding to the target (it could be in the form of isotope/fluorescent dye)

A

 PROBE LABELLING

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30
Q

3 BASIC METHODS FOR DNA PROBE LABELING:

A

 END LABELING
 NICK TRANSLATION
 RANDOM PRIMING

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31
Q

o 3 BASIC METHODS FOR DNA PROBE LABELING
- labeled nucleotides are added to the END OF THE PROBE using terminal transferase or T4 polynucleotide kinase

A

END LABELING

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32
Q

o 3 BASIC METHODS FOR DNA PROBE LABELING
- labeled nucleotides are incorporated into single-stranded breaks, or nicks, that are substrates for nucleotide addition by DNA polymerase

A

NICK TRANSLATION

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33
Q

o 3 BASIC METHODS FOR DNA PROBE LABELING
- generates new single-stranded versions of the probe with the incorporation of the labeled nucleotides

A

RANDOM PRIMING

34
Q

SOURCE:
- Transcription from a synthetic DNA temperature in vitro.
- Predesigned systems commercially available

A

RNA PROBES

35
Q

modified nucleic acids that has advantage of being resistant to nucleases that degrade DNA and RNA by breaking the phosphodiester backbone
(Synthetic and resistant to nucleases)
such as:
- PEPTIDE NUCLEIC ACIDS
- LOCKED NUCLEIC ACIDS
- UNLOCKED NUCLEIC ACIDS

A

OTHER NUCLEIC ACID PROBE TYPES

36
Q

Polyvinyl difluoride (PVDF) - proteins T
- products of a generalized response to a specific antigen.

A

POLYCLONAL ANTIBODIES

37
Q

Polyvinyl difluoride (PVDF) - proteins T
- products of a SINGLE clone of plasma B cells

A

MONOCLONAL ANTIBODIES

38
Q

Determine the specificity of the results

A

DESIGN & OPTIMAL HYBRIDIZATION OF PROBE

39
Q
  • 500-5000bp
  • GREATER SPECIFICITY — less affected by points.
  • mutations/polymorphisms
  • Difficult and expensive to synthesize.
A

LONGER PROBES

40
Q
  • less than 500bp
  • LESS SPECIFIC
  • Higher chance of being repeated randomly in unrelated religions of the genome.
  • Ideal for mutational analysis
A

SHORTER PROBES

41
Q
  • The procedure was first reported by Edwin Southern.
  • Detects specific DNA sequences.
A

SOUTHERN BLOTS

42
Q

SOUTHERN BLOTS TRANSFER METHODS:
- simple and relatively inexpensive; no requirements are required
- Driven by capillary movement of buffer from the soaked paper to the dry paper
- procedure is slow

A

CAPILLARY TRANSFER

43
Q

SOUTHERN BLOTS TRANSFER METHODS:
- uses ELECTRIC CURRENT to move the DNA transversely through the gel to the membrane.
- Used mostly for small fragments or proteins.

A

ELECTROPHORETIC TRANSFER

44
Q

SOUTHERN BLOTS TRANSFER METHODS:
- uses SUCTION and buffer recirculation to move the DNA out of the gel and onto the membrane;
- faster than capillary transfer for large DNA fragments
- Requires specialized equipment.

A

VACUUM TRANSFER

45
Q

SOUTHERN BLOTS
TYPES OF MEMBRANE?

A
  1. Nitrocellulose
  2. Nylon
  3. Cellulose modified with a dimethyl aminoethyl
  4. Carboxymethyl (CM) chemical group
  5. Polyvinyl difluoride (PVDF)
46
Q

SOUTHERN BLOTS (TYPES OF MEMBRANE)
- bind 70-150 µg of nucleic acids per cm squared
- High bonding capacity to nucleic acids & proteins

A

NITROCELLULOSE

47
Q
  • prevent the probe from binding to the nonspecific sites on the membrane surface
A

PREHYBRIDIZATION

48
Q

PREHYBRIDIZATION BUFFER COMPOSITION

A

 Denhardt solution (ficoll, polyvinyl pyrrolidone, bovine serum albumin)
 Salmon sperm DNA
 SDS sodium dodecyl acetate) , 0.01% may also be included with formamide

49
Q

o Membrane is exposed to the buffer at the optimal hybridization temperature for ?

A

30 mins several hours

50
Q

SOUTHERN BLOT APPLICATION

A
  1. ID of the single gene in a pool of DNA fragments.
  2. Gene mapping
  3. Analysis of genetic patterns of DNA
  4. Detection of specific DNA sequences in a genome
  5. Study of gene deletions, duplications, and mutations that cause various diseases
  6. Detection of genetic disease and cancer
  7. Detects the presence of a gene family in a genome
  8. DNA fingerprinting such as forensic tests, paternity testing, and sex determination
51
Q
  • Designed to investigate RNA structure and quantity:
    1. Levels of gene expression (transcription from DNA) and stability
    2. RNA structural abnormalities resulting from aberrations in synthesis/processing (alternative splicing)
A

NORTHERN BLOT APPLICATION

52
Q
  • IMMOBILIZED TARGET: protein
  • SDS-polyacrylamide gel electrophoresis (SDS-PAGE): resolves proteins according to molecular weight at 5%-20% concentrations.
  • Isoelectric focusing gels (IEF)/tube electrophoresis: resolves proteins according to charge.
  • OTHER MEMBRANES USED: PVDF and anion (DEAE) or cation (CM) exchange cellulose.
  • SIGNALS: chemiluminescent or color signals
A

WESTERN BLOT

53
Q

Combination of conditions under which the target is exposed to the probe

A

HYBRIDIZATION CONDITIONS, STRINGENCY

54
Q

o High hybridization temp + low concentration of salt in the buffers
o Probe will NOT BIND to its TARGET.

A

HIGH STRINGENCY CONDITIONS

55
Q

o Low hybridization temp + a high concentration of salt in the buffer
o Probe will NOT BIND to UNRELATED TARGETS.

A

LOW STRINGENCY CONDITIONS

56
Q

FACTORS AFFECTING STRINGENCY

A

o TEMP. of hybridization.
o SALT CONCENTRATION of the hybridization buffer.
o CONCENTRATION OF DENATURAT (formamide) in buffer.
o LENGTH AND NATURE OF THE PROBE SEQUENCE.

57
Q

Long probes with the higher percent of G and C bases WILL BIND under more stringent conditions than shorter probes with higher percent of A and G bases.

T OR F?

A

TRUE

58
Q

HOW TO ESTIMATE THE IDEAL HYBRIDIZATION CONDITIONS?

A

Calculate the melting temperature (T”m)” of the probe sequence.

59
Q

Hybridization are generally performed in

A

hybridization bags/glass cylinders

60
Q

RECOMMENDED VOLUME FOR HYBRIDIZATION BUFFER

A

approx. 10 mL/100 cm² of membrane surface area.

61
Q

Shorts probes (less than 20 bases) can hybridize in

A

1- 2 HOURS

62
Q

Long probes (greater than 1000 bases) can hybridize in

A

16 HOURS OR MORE

63
Q

___________ - Whether the probe has bound to the immobilized target.

A

DETECTION SYSTEM

64
Q

DETECTION SYSTEM
- Unbound probe is washed off and the blot is EXPOSED TO LIGHT- SENSITIVE FILM to detect the fragments that are hybridized to the radioactive probe.

A

DNA/ RNA Probe Labeled with Radioactive Phosphorus Atoms

65
Q

DETECTION SYSTEM
- Unbound probe is washed away and anti-digoxigenin antibody/streptavidin is added to bind labeled probe target complex

A

Indirect Nonradioactive Detection System

66
Q

Relies on microarrays, which are solid supports (e.g. glass slides/chips) containing an array of DNA & RNA probes that can hybridized with complementary nucleic acid sequences

A

ARRAY - BASED HYBRIDIZATION

67
Q

is a glass slide carrying hundreds to thousands of probes

A

MICROARRAY/DNA CHIP

68
Q
  • Applied to expression, mutation and amplification/deletion analyses.
  • Target DNA/RNA is deposited directly on the membrane by means of various devices (vacuum systems and pipette for few samples).
  • Performed on cloned plasmids, PCR products, selected mRNA selections
A

DOT/SLOT BLOTS

69
Q
  • Target is deposited in a CIRCLE or DOT.
  • Useful for MULTIPLE QUANTITATIVE ANALYSES where many targets are being compared (mutational screening)

DOT OR SLOT BLOT?

A

DOT BLOT

70
Q
  • Target is deposited in a OBLONG BAR
  • Useful for ACCURATE QUANTIFICATION by densitometry scanning.

DOT OR SLOT BLOT?

A

SLOT BLOT

71
Q
  • Type of hybridization analysis allows simultaneous study of a large number of targets.
  • APPROACHES:
    a) Macroassays
    b) Microassays
    c) High-density oligonucleotide arrays
    d) Microelectronic arrays
A

GENOMIC ARRAY TECHNOLOGY

72
Q

GENOMIC ARRAY TECHNOLOGY TYPE OF APPROACHES
- REVERSE DOT BLOT: immobilized probe is now effectively the target and the labeled specimen
o DNA, RNA, proteins are actually the probes
- DETECTION OF HYBRIDIZATION: radioactive /chemiluminescent signals
- READING OF HYBRIDIZATION: by eye or phosphorimager
- ANALYSIS: signal intensity from test “spots” was compared to control samples spotted on duplicate membranes.

A

MACROASSAY

73
Q

GENOMIC ARRAY TECHNOLOGY TYPE OF APPROACHES
- Glass substrates are used for the production of arrays.
- Improved spotting and has the ability to deposit very small target spots.
- Automated deposing systems (arrayers).
o Can place less than 80,000 spots on the glass substrate.
o Pen-type and ink-jet

A

MICROASSAYS

74
Q

GENOMIC ARRAY TECHNOLOGY TYPE OF APPROACHES
- High-density oligonucleotide array
- Deposit target by DNA synthesis directly on the glass/silicon support.
- Uses sequence information to design oligonucleotides at designated positions on the chip.
- Used for mutation and polymorphism analysis, DNA methylation analysis, and sequencing.

A

PHOTOLITHOGRAPHY TECHNIQUE

75
Q

PHOTOLITHOGRAPHY TECHNIQUE
- indicates amplification of the test sample locus.

A

EXCESS PURPLE LABEL

76
Q

PHOTOLITHOGRAPHY TECHNIQUE
- indicates deletion of the test sample locus.

A

EXCESS BLACK LABEL

77
Q

PHOTOLITHOGRAPHY TECHNIQUE
- indicates equal test and reference DNA.

A

NEUTRAL OR GRAY

78
Q
  • Probes are immobilized on beads, allowing hybridization of the targets in the bead suspension.
  • Multiple suspensions can be tested simultaneously.
  • Beads are color-coded with a particular shade of red fluorescent dye.
  • Sample is labeled with a green dye.
  • Used for protein and nucleic acid targets.
  • Available for infectious diseases and tissue typing.
A

BEAD ARRAY TECHNOLOGY

79
Q
  • Neither the probe nor the target is immobilized.
  • Probes and targets bind in solution, followed by resolution of the bound products.
  • To measure mRNA expression
A

SOLUTION HYBRIDIZATION

80
Q

SOLUTION HYBRIDIZATION VERSION
- labeled probe probe is hybridized to the target sample in solution.

A

RNAseprotection (S1 mapping)

81
Q

SOLUTION HYBRIDIZATION VERSION
- uses 2 probes (capture probe and detection probe.

A

Capture of DNA probe: RNA target hybrids on a solid support/beads

MOL. BIO LEC (14 decks)

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