Module 6: Detection and Analysis of Nucleic Acid

Question 1

Macromolecules that exists as polymers
called polynucleotides.

Answer 1

Nucleic acid

Question 2

Formation of a nucleotide by adding ____ group to a nucleoside

Answer 2

phosphate groups

Question 3

phenol and chloroform/isoamyl alcohol
(25:24:1) are mixed with an equal
volume of samples by vortexing

Answer 3

Organic purification

Question 4

involves the incubation of nuclei with only proteinase K at 65°C

Answer 4

Inorganic Purification

Question 5

movement of DNA or RNA in response to an electric field

Answer 5

Electrophoresis Separation

Question 6

DNA are reduced to fragments by digestion with ____.

Answer 6

restriction enzymes

Question 7

are small molecules and no digestion is required prior to electrophoresis.

Answer 7

RNA

Question 8

Routinely uses Gel electrophoresis.
Uses TAE or TBE.

Answer 8

Molecular separation of DNA

Question 9

interaction between single stranded nucleic acid to form a (duplex) double stranded molecules based on complementary base
pairing of the sequences.

Answer 9

Nucleic Acid Hybridization

Question 10

process of recombination of two non-labeled strands into a stable double stranded structure.

Answer 10

annealing

Question 11

If one strand has a marker, a hybrid is formed between labeled and an unlabeled strand.

Answer 11

hybridization

Question 12

labeled strand is called the ____ and the product is called ____

Answer 12

probe; hybrid

Question 13

sample and probe in a solution

Answer 13

Liquid or Solution Phase Hybridization

Question 14

e.g. Digene Hybrid-Capture 2, Digene Corp. USA

Answer 14

Liquid/Solution Phase Hybridization

Question 15

Hybridization assay formats for detection of HPV

Answer 15

Liquid or Solution Phase

Question 16

occurs in biphasic environment

Answer 16

Solid Support Hybridization

Question 17

solid phase in solid support hybridization

Answer 17

sample

Question 18

liquid phase in solid support hybridization

Answer 18

probe

Question 19

e.g. dot or blot

Answer 19

Solid Support Hybridization

Question 20

What are the two Hybridization assay formats

Answer 20

Liquid or Solution Phase Hybridization
Solid Support Hybridization

Question 21

described by E.M. Southern in 1975, detect specific DNA sequences and gene mutations

Answer 21

Southern Blot

Question 22

Blotting techniques in which examples are electrophoretic separation, transferring to solid support nitrocellulose and hybridization

Answer 22

Southern blot

Question 23

Blotting technique that uses RNA sample

Answer 23

Northern Blot

Question 24

RNA is extacted, digested, electrophoresed, blotted and probed.

not routinely used in clin lab because RNA is not stable

Answer 24

Northern blot

Question 25

in northern blot technique, how many minutes and in what temp is rna denatured?

Answer 25

15 min of 55 C heating

Question 26

used to identify proteins separated by PAGE

Answer 26

Western Blot

Question 27

confirmatory for HIV and used in the diagnosis of Lyme Disease

Answer 27

Western blot

Question 28

membranes used in western blotting

Answer 28

- nitrocellulose - nylon - PVDF or Polyvinylidene difluoride

Question 29

probe molecule in southern and northern blotting

Answer 29

DNA

Question 30

western blotting probe molecule

Answer 30

antibody

Question 31

south-western blotting probe molecule

Answer 31

dsDNA

Question 32

Results from a variable number of tandem repeats (VNTR) in a short DNA FRAGMENT. used in FORENSIC DIAGNOSIS, DONOR TRANSPLANTS.

Answer 32

RESTRICTION FRAGMENT LENGTH POLYMORPHISM

Question 33

In RFLP, restriction fragments are separated on gel and analyzed by ____ blotting techniques.

Answer 33

southern

Question 34

*amplified nucleic acids, whether an RNA or DNA are referred to as

Answer 34

amplicons

Question 35

patented the PCR method in 1985

Answer 35

Kary Mullis

Question 36

target amplification method if the nucleic acid of interest is RNA rather than DNA, conversion of RNA to DNA

Answer 36

reverse-transcriptase PCR

Question 37

TA method which uses numerous primers for different targets in the same mixture

Answer 37

Multiplex PCR

Question 38

target amplificationa nd detection steps occur simultaneously and PCR product is detected using fluoresent dyes

Answer 38

Real-Time PCR

Question 39

uses probes or pieces of DNA or RNA that are labelled with a detectable molecule, such as biotin, dyes, or radioactive isotopes. These probes bind complementary to the target.

Answer 39

Probe Amplification Methods

Question 40

examples of probe amplification methods

Answer 40

ligase chain reaction, cycling probe technology, cleavase invader

Question 41

what are examples of amplification methods

Answer 41

target amplification probe amplification signal amplification

Question 42

Relies on specific recognition and cleavage of particular DNA structures by flap endonuclease-I family of DNA polymerases

Answer 42

Cleavase/Invader Technology

Question 43

two primers are designed that hybridize target sequence in an overlapping manner.

Answer 43

Cleavase/ Invader Technology

Question 44

uses a stimuli to generate a signal, where the signal is proportional to the amount of the target sequence present in the clinical specimen assays

Answer 44

Signal Amplification Methods

Question 45

examples are Branched DNA and Hybrid capture Assay

Answer 45

Signal amplification methods

Question 46

a fully automated method, dsDNA is target of exponential amplification, amplifies target nucleic acid without use of thermocycler

Answer 46

Strand displacement amplification

Question 47

an isothermal assay, targets either DNA or RNA ; RNA is the amplified product, detection of M. tuberculosis

Answer 47

TMA or Transcription Mediate Amplification

Question 48

similar to TMA but only RNA is targeted for amplification; detection of HIV and CMV

Answer 48

NASBA or Nucleic Acid Sequence-Based Amplification

Question 49

uses a collection of spots attached to a solid support that is capable of quantitating hundreds or even thousands of genes in a cell or a tissue simultaneously

Answer 49

Microarray or DNA Chip technology

Question 50

separation of dsDNA to ssDNA and breaking of H bonds between base pairs

Answer 50

DENATURATION

Question 51

between individual nucleotides or phosphate backbone; breaks in the backbone of DNA molecule either dsDNA or ssDNA

Answer 51

DEGRADATION

Question 52

2 Causes of DNA Degradation

Answer 52

1. Using very old DNA samples 2. Using DNA extracted from formalin-fixed paraffin embedded samples

Question 53

short term storage in weeks for DNA sample

Answer 53

4 C in Tris-EDTA

Question 54

medium-term storage in months of DNA

Answer 54

-80 C in Tris-EDTA

Question 55

Long-term storage in years under ethanol of DNA

Answer 55

-80 C as a precipitate

Question 56

long term storage of DNA in decades

Answer 56

-164 C or dried

Question 57

steps to prevent DNA degradation

Answer 57

- correct handling and storage - perform extractions at 40C on ice or in the cold - inhibit nuclease activity - store purified DNA correctly

Question 58

addition of phosphate groups is via

Answer 58

phosphoric anhydride linkage

Question 59

examples of methods to determine nucleic acid purity and yield

Answer 59

- DNA Spectrophotometry - Nanodrop Spectrophotometry - Fluorometric Methods - Real-Time PCR

Question 60

Methods for Nucleic Acid Analyses

Answer 60

- Electrophoresis - Hybridization Assay - DNA Sequencing - Polymorphism based - Amplification Techniques

Question 61

AGE means

Answer 61

Agarose Gel Electrophoresis

Question 62

PAGE

Answer 62

Polyacrylamide Gel Electrophoresis

Question 63

SDS-PAGE

Answer 63

Sodium dodecyl-sulfate Polyacrylamide gel Electrophoresis

Question 64

DGGE

Answer 64

Denaturing gradient gel electrophoresis (DGGE)

Question 65

TAE means

Answer 65

Tris-acetate-EDTA

Question 66

TBE

Answer 66

Tris-Borate-EDTA

Question 67

short nucleotide sequence complementary to a specific DNA sequence and initiate DNA replication

Answer 67

Primers

Question 68

provides a free 3'-OH for DNA polymerase to start synthesis of chain

Answer 68

primers

Question 69

use to target a particular sequence of complementary DNA or RNA ; can be labeled with radionuclide (P32), enzyme, and biotin

Answer 69

Probes