MIDTERM LEC 3

Question 1

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

Answer 1

NUCLEIC ACID/PROTEIN ANALYSIS

Question 2

NUCLEIC ACID/PROTEIN ANALYSIS 3 MAIN METHODS

Answer 2

1. Restriction enzyme mapping of DNA
2. CRISPR enzyme systems
3. Hybridization technology

Question 3

• 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.

Answer 3

Restriction enzyme mapping of DNA

Question 4

commonly used endonuclease in restriction enzyme mapping of DNA

Answer 4

TYPE II ENDONUCLEASE

Question 5

*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

Answer 5

Restriction Fragment Length Polymorphisms (RFLPs)

Question 6

RESTRICTION MAPPING USES/APPLICATIONS

Answer 6

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.

Question 7

• 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.

Answer 7

CRISPR enzyme systems

Question 8

CRISPR stands for

Answer 8

CLUSTERED REGULARLY INTERSPACED REPEATS

Question 9

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

Answer 9

dsDNA

Question 10

CRISPR enzyme systems TYPES:
Type III - target ______

Answer 10

ssDNA & RNA

Question 11

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

Answer 11

Hybridization TECHNOLOGY

Question 12

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

Answer 12

HYBRIDIZATION

Question 13

IN HYBRIDIZATION TECHNIQUE:
- First strand is _________

Answer 13

UNLABELED

Question 14

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

Answer 14

Southern blot

Question 15

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

Answer 15

Northern blot

Question 16

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

Answer 16

WESTERN BLOT

Question 17

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

Answer 17

Southwestern blot

Question 18

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

Answer 18

Eastern blot

Question 19

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

Answer 19

Far-eastern blot

Question 20

Southern blot – procedure was first reported by

Answer 20

Edwin Southern

Question 21

• 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.

Answer 21

PROBE

Question 22

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

Answer 22

Nucleic Acid (NA) Probes (DNA probes)

Question 23

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

Answer 23

Nucleic Acid (NA) Probes (RNA probes)

Question 24

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

Answer 24

Protein Probes

Question 25

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

Answer 25

SEQUENCE OF INTEREST

Question 26

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

Answer 26

95°C, 10 – 15min

Question 27

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

Answer 27

75°C; 5-6 min

Question 28

 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

Answer 28

DNA PROBES

Question 29

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

Answer 29

 PROBE LABELLING

Question 30

3 BASIC METHODS FOR DNA PROBE LABELING:

Answer 30

 END LABELING  NICK TRANSLATION  RANDOM PRIMING

Question 31

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

Answer 31

END LABELING

Question 32

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

Answer 32

NICK TRANSLATION

Question 33

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

Answer 33

RANDOM PRIMING

Question 34

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

Answer 34

RNA PROBES

Question 35

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

Answer 35

OTHER NUCLEIC ACID PROBE TYPES

Question 36

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

Answer 36

POLYCLONAL ANTIBODIES

Question 37

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

Answer 37

MONOCLONAL ANTIBODIES

Question 38

Determine the specificity of the results

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DESIGN & OPTIMAL HYBRIDIZATION OF PROBE

Question 39

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

Answer 39

LONGER PROBES

Question 40

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

Answer 40

SHORTER PROBES

Question 41

* The procedure was first reported by Edwin Southern. * Detects specific DNA sequences.

Answer 41

SOUTHERN BLOTS

Question 42

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

Answer 42

CAPILLARY TRANSFER

Question 43

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.

Answer 43

ELECTROPHORETIC TRANSFER

Question 44

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.

Answer 44

VACUUM TRANSFER

Question 45

SOUTHERN BLOTS TYPES OF MEMBRANE?

Answer 45

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

Question 46

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

Answer 46

NITROCELLULOSE

Question 47

- prevent the probe from binding to the nonspecific sites on the membrane surface

Answer 47

PREHYBRIDIZATION

Question 48

PREHYBRIDIZATION BUFFER COMPOSITION

Answer 48

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

Question 49

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

Answer 49

30 mins several hours

Question 50

SOUTHERN BLOT APPLICATION

Answer 50

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

Question 51

- 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)

Answer 51

NORTHERN BLOT APPLICATION

Question 52

- 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

Answer 52

WESTERN BLOT

Question 53

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

Answer 53

HYBRIDIZATION CONDITIONS, STRINGENCY

Question 54

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

Answer 54

HIGH STRINGENCY CONDITIONS

Question 55

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

Answer 55

LOW STRINGENCY CONDITIONS

Question 56

FACTORS AFFECTING STRINGENCY

Answer 56

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.

Question 57

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?

Answer 57

TRUE

Question 58

HOW TO ESTIMATE THE IDEAL HYBRIDIZATION CONDITIONS?

Answer 58

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

Question 59

Hybridization are generally performed in

Answer 59

hybridization bags/glass cylinders

Question 60

RECOMMENDED VOLUME FOR HYBRIDIZATION BUFFER

Answer 60

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

Question 61

Shorts probes (less than 20 bases) can hybridize in

Answer 61

1- 2 HOURS

Question 62

Long probes (greater than 1000 bases) can hybridize in

Answer 62

16 HOURS OR MORE

Question 63

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

Answer 63

DETECTION SYSTEM

Question 64

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.

Answer 64

DNA/ RNA Probe Labeled with Radioactive Phosphorus Atoms

Question 65

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

Answer 65

Indirect Nonradioactive Detection System

Question 66

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

Answer 66

ARRAY - BASED HYBRIDIZATION

Question 67

is a glass slide carrying hundreds to thousands of probes

Answer 67

MICROARRAY/DNA CHIP

Question 68

- 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

Answer 68

DOT/SLOT BLOTS

Question 69

- 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?

Answer 69

DOT BLOT

Question 70

- Target is deposited in a OBLONG BAR - Useful for ACCURATE QUANTIFICATION by densitometry scanning. DOT OR SLOT BLOT?

Answer 70

SLOT BLOT

Question 71

- 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

Answer 71

GENOMIC ARRAY TECHNOLOGY

Question 72

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.

Answer 72

MACROASSAY

Question 73

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

Answer 73

MICROASSAYS

Question 74

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.

Answer 74

PHOTOLITHOGRAPHY TECHNIQUE

Question 75

PHOTOLITHOGRAPHY TECHNIQUE - indicates amplification of the test sample locus.

Answer 75

EXCESS PURPLE LABEL

Question 76

PHOTOLITHOGRAPHY TECHNIQUE - indicates deletion of the test sample locus.

Answer 76

EXCESS BLACK LABEL

Question 77

PHOTOLITHOGRAPHY TECHNIQUE - indicates equal test and reference DNA.

Answer 77

NEUTRAL OR GRAY

Question 78

- 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.

Answer 78

BEAD ARRAY TECHNOLOGY

Question 79

- 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

Answer 79

SOLUTION HYBRIDIZATION

Question 80

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

Answer 80

RNAseprotection (S1 mapping)

Question 81

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

Answer 81

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