NUCLEIC ACID HYBRIDIZATION

Question 1

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

Answer 1

nucleic acid hybridization

Question 2

uses of nucleic acid hybridization

Answer 2

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

Question 3

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

Answer 3

Probe

Question 4

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

Answer 4

TARGET

Question 5

PROBE TYPE: DNA
Early method of production:

Answer 5

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

Question 6

T OR F???
* Shorter probes are more specific

• Longer probes are better for
  mutational analyses

Answer 6

F
* Longer probes are more specific
* Shorter probes are better for
mutational analyses

Question 7

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

Answer 7

PROBE TYPE: RNA

Question 8

LABELING THE PROBE

END Labeling
Labeled molecules added to the end using ___

Answer 8

terminal transferase
or T4 polynucleotide
kinase

Question 9

Introduction of nucleotides with
radioactive phosphorus (32P), sulfur (35S), or nitrogen to the probe

Answer 9

Radioisotopes

Question 10

Non Radioactive labels

• Most common: ___ and
  ___ attached to a UTP or CTP
• Can also be ___

Answer 10

biotin and digoxygenin (1)
fluorescent dyes (FISH) (2)

Question 11

Non Radioactive labels
Detection:
1. Washing off of unbound probe
2. ____ or ____ are conjugated to ____ or ____
3. Washing off of unbound conjugate
4. Bathing in substrate (dioxetane
or tetrazolium dye)

Answer 11

Antidioxygenin (dioxygenin) or streptavidin (biotin) (1)
alkaline phosphatase (AP) or horseradish peroxidase (HRP) (2)

Question 12

Southern Blot
* Target: DNA
* Probe: ____
* First done by ____

Answer 12

Nucleic acid (1)
Edwin Southern (2)

Question 13

Southern Blot
unsa tung 1-6????

Answer 13

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

Question 14

Restriction enzyme digestion

____ have sequence-specific activity with 4-6 bp palindromic recognition sites

Answer 14

Restriction endonucleases

Question 15

Resolution of the cut fragments are done by ___

Answer 15

gel electrophoresis

Question 16

RESOLUTION
* COMMON PROBLEMS:
* Large aggregates near the top –

Answer 16

incomplete digestion

Question 17

RESOLUTION
* Smear in the lower region –

Answer 17

degraded DNA

Question 18

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

Answer 18

Denaturation

Question 19

Can be done directly using ___ or using ___ for depurination prior to denaturation (for >500 bp fragments)

Answer 19

NaOH or using HCl

Question 20

“Transfer” of denatured DNA to a solid substrate (usually nitrocellulose or nylon)

Answer 20

Blotting

Question 21

T OR F?

Blotting

CAPILLARY TRANSFER
* Gel is placed on the bottom of the high salt buffer (10X saline sodium citrate)

Answer 21

F : Gel is placed on top of high salt buffer

Question 22

T OR F?
Blotting is faster than capillary transfer

Answer 22

T

Question 23

Immobilization of the DNA is done by baking in a ___ or by ___ to the membrane

Answer 23

vacuum oven or by UV-crosslinking

Question 24

• Target: RNA
• Probe: Nucleic acid

Answer 24

Northern Blot

Question 25

Degree if homology between the probe and the NA

Answer 25

STRINGENCY
(not sure if mao ang meaning)

Question 26

Temperature formula

Answer 26

𝑇𝑚 = 81.5 deg 𝐶 + 16.6 log 𝑀 + 0.41 %𝐺𝐶 − 0.61 % 𝑓𝑜𝑟𝑚𝑎𝑚𝑖𝑑𝑒 − (600/𝑛)

Question 27

Sequence complexity formula

Answer 27

𝐶𝑜𝑡 = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐷𝑁𝐴 × 𝑡𝑖𝑚𝑒 𝑓𝑜𝑟 𝑟𝑒𝑎𝑛𝑛𝑒𝑎𝑙𝑖𝑛g

Question 28

Incubation system
✓ The PROBE is the ___ during hybridization

Answer 28

limiting reagent

Question 29

Recommended hybridization buffer volume: ___ of the membrane surface area

Answer 29

10 mL/100 cm2

Question 30

Incubation within a ___ or___ maintains optimal
hybridization temp

Answer 30

sealed bag in a water bath or capped glass cylinders in rotary ovens

Question 31

Can analyze larger numbers of
samples at the same time

Answer 31

Macroarrays

Question 32

Macroarrays

Uses treated glass (“____”) instead of nitrocellulose or nylon

Answer 32

chip

Question 33

Northern Blot
Differences with Southern:
* RNase-free environment must
be maintained

Answer 33

T

Question 34

Northern Blot
Differences with Southern:
* 15X SSC vs 10X SSC in Southern

Answer 34

F : 20X SSC vs 10X SSC

Question 35

What is the goal of blotting?

Answer 35

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