Nucleic Acids & Protein Characterization

Question 1

What are nucleases?

Answer 1

Enzymes that degrade DNA molecules by breaking the phosphodiester bonds between nucleotides.

Question 2

What are the two types of nucleases?

Answer 2

1. Exonuclease
   
   2. Endonuclease

Question 3

What is the function of exonucleases?

Answer 3

Exonucleases remove nucleotides one at a time from the end of a DNA molecule.

Question 4

How do exonucleases act on DNA strands?

Answer 4

• Some attack from 5’ and hydrolyze from the 5’ to 3’ end.
• Others attack 3’ and hydrolyze from the 3’ to 5’ end.

Question 5

What are DNases and RNases?

Answer 5

• DNases degrade DNA.
• RNases degrade RNA.

Question 6

What is RNase H?

Answer 6

An enzyme that specifically cleaves the RNA strand of a DNA-RNA hybrid.

Question 7

What determines the type of nucleotide produced when a nuclease hydrolyzes a phosphodiester bond?

Answer 7

It depends on the ester bond attacked, generating either 5’ nucleotides or 3’ nucleotides.

Question 8

What determines a nuclease’s strand preference?

Answer 8

Nucleases may be specific for single-stranded, double-stranded, or both types of DNA.

Question 9

Which exonuclease removes nucleotides from both strands of a double-stranded DNA?

Answer 9

Bal31

Question 10

Which exonuclease degrades only one strand of a double-stranded DNA? leaving a single stranded DNA as the product

Answer 10

Exonuclease III

Question 11

What is the key function of endonucleases?

Answer 11

Endonucleases hydrolyze internal bonds within a polynucleotide chain (break in the middle)

Question 12

What is the difference between 5’ endonuclease and DNase I?

Answer 12

• 5’ Endonuclease – Cleaves only single-stranded DNA.
• DNase I – Cleaves both single- and double-stranded DNA.

Question 13

What is the key feature of Taq DNA polymerase?

Answer 13

• It is thermostable, making it suitable for PCR (Polymerase Chain Reaction).
• enzyme of bacterium thermus aquaticus

Question 14

What is the function of Reverse Transcriptase?

Answer 14

It synthesizes complementary DNA (cDNA) from an RNA template in RT-PCR.

Question 15

What enzyme removes the 5’ phosphate group from DNA?

Answer 15

Alkaline Phosphatase (from E. coli, calf intestinal tissue, or arctic shrimp).

Question 16

What enzyme adds a phosphate group onto free 5’ termini of DNA?

Answer 16

Polynucleotide Kinase (from E. coli infected with T4 phage)

Question 17

What enzyme adds deoxyribonucleotides to the 3’ terminus of DNA molecules?

Answer 17

Terminal Deoxynucleotidyl Transferase (from calf thymus tissue).

Question 18

What enzyme repairs single-stranded breaks in DNA?

Answer 18

DNA Ligase — important in DNA replication and repair

Question 19

What additional function does DNA Ligase have aside from repairing single-strand breaks?

Answer 19

It can join two DNA fragments or ligate the ends of the same DNA molecule.

Question 20

It is the formation of hydrogen bonds between two complementary nucleic acid strands.

Answer 20

Nucleic Acid Hybridization?

Question 21

A labeled nucleic acid strand that binds to a complementary strand to detect the presence of specific sequences.

Answer 21

probe

Question 22

Why is a probe important in hybridization?

Answer 22

It allows the detection and measurement of nucleic acid content in an unknown sample.

Question 23

What is the purpose of a Southern Hybridization assay?

Answer 23

Used to identify homologous sequences in genomic DNA and assist in gene mapping through restriction mapping.

Question 24

What are examples of Hybridization Assays?

Answer 24

1. Southern Hybridization (DNA)
   
   2. Northern Hybridization (RNA)
   3. Dot/Blot Hybridization
   4. Microarray
   5. Fluorescent In Situ Hybridization (FISH)

Question 25

identifies homologous sequences in genomic DNA

Answer 25

Southern Hybridization (DNA)

Question 26

southern hybridization facilitates in ___ and detects ___

Answer 26

Facilitates gene mapping and detects restriction fragment length polymorphisms (RFLPs)

Question 27

when and why is rna denatured

Answer 27

rna must be denatured before and during electrophoresis to prevent secondary structure formation

Question 28

denaturation step for rna: heat to ___ with the presence of __ and __

Answer 28

heat to 55C with formaldehyde and formamide

Question 29

during electrophoresis what does the formaldehyde do?

Answer 29

formaldehyde in the gel prevents secondary structure reformation

Question 30

in **post electrophoresis**: RNA is **transferred** to a nylon membrane via ___ or ___ and **fixed** by ___ or ___

Answer 30

transferred via capillary or vacuum blotting and fixed by UV light or baking at 80C

Question 31

not recommended during electrophoresis

Answer 31

ethidium bromide

Question 32

why is ethidium bromide not recommended during electrophoresis

Answer 32

it decreases hybridization signal

Question 33

how do you prep sample in simplified northern blot

Answer 33

mix RNA sample with denaturing agents like MOPS, formaldehyde, and loading dye

Question 34

how do you denature in simplified northern blot

Answer 34

quench with ice

Question 35

electrophoresis in simplified northern blot

Answer 35

run on a 1.2% agarose formaldehyde gel at 70V for 3.5 hours with buffer recirculation

Question 36

RLFP (Restriciton Fragment Length Polymorphism) results from ___ in a DNA segment

Answer 36

VNTR (Variable Number Tandem Repeats)

Question 37

RFLP is used in?

Answer 37

- DNA fingerprinting - paternity testing - genetic disease marker

Question 38

RFLP analysis process

Answer 38

1. Genomic DNA is collected and digested with a restriction enzyme 2. DNA fragments are separated by gel electrophoresis 3. The gel undergoes Southern blot analysis for fragment detection

Question 39

RFLP analysis: ___ is collected and digested with a ___

Answer 39

genomic dna is collected and digested with a restriction enzyme

Question 40

RFLP analysis: DNA are separated by ___

Answer 40

DNA fragments are separated by gel electrophoresis

Question 41

RFLP analysis: the gel undergoes __ for fragment detection

Answer 41

southern blot analysis

Question 42

can amplify small DNA amounts for faster RFLP analysis usually in **2-3H** therefore more samples can be analyzed in a shorter time

Answer 42

PCR

Question 43

PCR can amplify small DNA amounts for faster RFLP analysis , this is called the?

Answer 43

CAPS (Cleaved Amplified Polymorphic Sequence Assay)

Question 44

evaluates hundreds to thousand of DNA regions across the genome without needing prior sequence knowledge

Answer 44

AFLP (Amplified Fragment Length Polymorphism)

Question 45

AFLP is useful for ___ that lack complete genome sequences and where other types of genome-wide markers such as **single nucleotide polymorphisms (SNPs)** and **microsatellites** are difficult to obtain

Answer 45

non model species

Question 46

AFLP are ideal for identifying ____ when ___ and __ are challenging to obtain

Answer 46

ideal for identifying genetic variations when SNPs and micro satellites are challenging to obtain

Question 47

what are the 3 DNA modifying enzymes

Answer 47

- alkaline phosphatase - polynucleotide kinase - terminal deoxynucleotidyltransferaseadds

Question 48

what are DNA MODIFYING ENZYMES

Answer 48

modify DNA molecules by addition or removal of specific chemical groups