Nucleic Acid Hybridization

Question 1

Takes advantage of the bonding of single-stranded nucleic acids with their complementary sequences
to identify specific DNA sequences.

Answer 1

NA HYBRIDIZATION

Question 2

Hybrid strands can be

Answer 2

DNA-DNA, DNA-RNA, or RNA-RNA

Question 3

NA HYBRIDIZATION

USES:

Answer 3

• Detection of specific sequences in a mixture of DNA fragments or gDNA
• Determination of base sequence similarities and changes

Question 4

Single-stranded fragment of nucleic acid attached to a signal-producing moiety to identify sequence/s of interest

Answer 4

Probe

Question 5

probe

Labelled with a ___ or ____

Answer 5

radioisotope or fluorescent molecule

Question 6

Denatured DNA or RNA that has good complementary to the probe for identification

Answer 6

Target

Question 7

Bound to a nylon membrane or another solid surface

Answer 7

Target

Question 8

Probe type: DNA
1. Cloning into a plasmid
2. Isolation by RE digestion
3. Gel purification
4. Labeling
5. Denaturation (heating alone or with formamide)

Answer 8

Early method of production

Question 9

Probe type: DNA
• Isolation from viral genomes
• In vitro organic synthesis (short, oligomeric probes)
• PCR (for production of large amounts)

Answer 9

New methods of production

Question 10

Probe type: DNA

•_____ probes are more specific
•_____ probes are better for mutational analyses (can detect single base changes); has high background noise otherwise

• For both DNA and RNA probes, sequence matters (e.g. internal complements, and high GC content)

Answer 10

Longer

Shorter

Question 11

• Made by in vitro transcription from
DNA (from plasmid template or PCR amplicons)

Answer 11

PROBE TYPE: RNA

Question 12

Probe

• Has equal or greater binding affinity

Answer 12

RNA probe

Question 13

Probe

• More sensitive

Answer 13

RNA probes

Question 14

Production:
1. Cloning into plasmid
2. Linearization of recombinant vector
3. Transcription from promoter
• Normal - Southern
• Antisense - Northern
4. Labeling
5. Removal of DNA template

Answer 14

RNA probe

Question 15

Must be done so the probe generates a detectable signal

Answer 15

Labeling the probe

Question 16

Labeled molecules added to the end using terminal transferase or T4 polynucleotide kinase

Answer 16

END Labeling

Question 17

End labeling

Labeled molecules added to the end using (2)

Answer 17

terminal transferase
T4 polynucleotide kinase

Question 18

Labeled nucleotides added into single-stranded breaks that become substrates for DNA polymerase to extend the nick

Answer 18

Nick Translation

Question 19

Generation of new single-stranded version of the probe with labeled nucleotides

Answer 19

Random priming

Question 20

• Introduction of nucleotides with radioactive phosphorus (32P), sulfur
(355), or nitrogen to the probe

Answer 20

Radioisotope

Question 21

• Unbound probes get washed off

• Blots are exposed to light-sensitive film to detect the hybridized fragments

Answer 21

Radioisotope

Question 22

Radioisotope

• Introduction of nucleotides with ( either 3) to the probe

Answer 22

radioactive phosphorus (32P)

sulfur (35S)

nitrogen

Question 23

No longer favored!
• Hazardous to humans and the environment
• Half-lives are short and thus, experiments must be done quickly

Answer 23

Radioisotope

Question 24

• Based on indirect detection of tagged nucleotides incorporated or added to the probe

Answer 24

Non Radioactive labels

Question 25

Non radioactive labels • Most common: (2) • Can also be (1)

Answer 25

biotin digoxygenin attached to a UTP or CTP fluorescent dyes (FISH)

Question 26

Labels Detection: 1. Washing off of unbound probe 2. _____ or _____ are conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP) 3. Washing off of unbound conjugate 4. Bathing in substrate (dioxetane or tetrazolium dye)

Answer 26

Antidioxygenin (dioxygenin) or streptavidin (biotin)

Question 27

Southern blot • Target: • Probe: • First done by____

Answer 27

DNA Nucleic acid Edwin Southern

Question 28

• Mostly used to study gene structure and detection in a sample

Answer 28

Southern blot

Question 29

Southern blot procedure

Answer 29

1. RE digestion/cutting 2. Resolution 3. Denaturation of fragments 4. Blotting (Transfer) 5. Probing 6. Detection

Question 30

•_________ have sequence-specific activity with____ bp palindromic recognition sites • Cut up nucleic acids in a predictable manner that can be mapped (restriction site mapping) to determine the linear order of sequences on a strand

Answer 30

Restriction endonucleases 4-6

Question 31

Under proper reaction conditions, restriction endonuclease are highly specific • Number and location of restriction sites vary among individuals (restriction fragment length polymorphisms, RFLP) •_____altered specificity in nonstandard reaction conditions

Answer 31

Star activity

Question 32

Digestion is carried out for an extended time (_____hours) to allow complete cutting of the sites

Answer 32

1-3 hrs

Question 33

• Resolution of the cut fragments are done by______ • Larger fragments require longer runs at_____ voltage

Answer 33

gel electrophoresis lower voltage

Question 34

Resolution • COMMON PROBLEMS: • Large aggregates near the top -_____ • Smear in the lower region -____

Answer 34

incomplete digestion degraded DNA

Question 35

• Preparation before blotting

Answer 35

Denaturation

Question 36

Denaturation • Can be done directly using ____ or _____for depurination prior to denaturation (for >500 bp fragments)

Answer 36

NaOH or using HCl

Question 37

• Double-stranded DNA are separated into their single-stranded form for later probing

Answer 37

Denaturation

Question 38

• "Transfer" of denatured DNA to a solid substrate (usually nitrocellulose or nylon)

Answer 38

Blotting

Question 39

• Goal: move DNA from the gel to the membrane for probing

Answer 39

Blotting

Question 40

Used in blotting Usually (2)

Answer 40

nitrocellulose or nylon

Question 41

Blotting CAPILLARY TRANSFER • Gel is placed on top of ____buffer (10X saline sodium citrate) • Absorbent paper stacked on top Movement by capillary action • Usually left overnight (~____h)

Answer 41

high salt 16h

Question 42

• Gel is placed on top of high salt buffer (10X saline sodium citrate) • Absorbent paper stacked on top Movement by capillary action • Usually left overnight (~16h)

Answer 42

CAPILLARY TRANSFER

Question 43

• Use of electric current to carry DNA • Similar to AGE but ran vertically • Faster than capillary transfer

Answer 43

ELECTROPHORETIC TRANSFER

Question 44

• Use of suction to move DNA in a recirculating buffer Also faster than capillary (2-3h) Avoids discontinuous transfer d/t air

Answer 44

VACUUM TRANSFER

Question 45

Blotting • Immobilization of the DNA is done by baking in a (2) to the membrane

Answer 45

vacuum oven or by UV-crosslinking

Question 46

• Prevents the probe from binding nonspecifically • Incubation of membrane in the same buffer as the probe prior to introduction

Answer 46

Prehybridization step

Question 47

Northern Blot • Target: • Probe: • Modification of the Southern blot

Answer 47

RNA Nucleic acid

Question 48

• Studying RNA structure and gene expression, mutational analyses

Answer 48

Northern blot

Question 49

• RNase-free environment must be maintained

Answer 49

Northern blot

Question 50

• AGE ran under denaturing conditions - no separate denaturation step • 20X SSC vs 10X SSC in Southern

Answer 50

Northern blot

Question 51

Temperature NOTES: • Tm of_____ probes is higher • RNA:DNA hybrids increase Tm by___ • RNA:RNA hybrids increase Tm by___ • Optimal hybridization temp is ____below Tm

Answer 51

RNA probes 10-15°C 20-25°C ~5°C

Question 52

Incubation system • The ______ is the limiting reagent during hybridization • Sensitivity increases with probe concentration • Keep volume of hybridization solution low

Answer 52

PROBE

Question 53

THUS: Recommended hybridization buffer volume: _______of the membrane surface area Incubation within a_____ in a water bath or capped glass cylinders in rotary ovens maintains optimal hybridization temp

Answer 53

10 mL/100 cm sealed bag

Question 54

• Can analyze larger numbers of samples at the same time

Answer 54

Macroarrays DOT/SLOT Blots

Question 55

• LIMITATIONS: • Area of substrate material and membrane • Volume of hybridization solution • Testing for only one gene product

Answer 55

Macroarrays DOT/SLOT Blots

Question 56

• Uses treated glass ("chip") instead of nitrocellulose or nylon • Tens of thousands of targets can be screen in a small area using only small amounts of sample simultaneously • Makes use of automated arrayers

Answer 56

Microarrays