NUCLEIC ACID AMPLIFICATION TECHNIQUES

Question 1

the very first amplification method

Answer 1

PCR - polymerase chain reaction

Question 2

Nucleic acid (NA) amplification methods fall
into three categories:

Answer 2

target amplification
probe amplification
signal amplification

Question 3

Involves making copies of a target
sequence to such a level (in millions
of copies) that they can be detected
in vitro.

Answer 3

target amplification

Question 4

This is analogous to growing cells in
the culture and allowing the cells to
replicate their nucleic acids, as well
as themselves

Answer 4

target amplification

Question 5

The difference between
microbiology and molecular
biology amplification is

Answer 5

in microbiology - amplification is days to months

in target amplification minutes to hours

Question 6

–The first prototypical (specific)
method for amplifying target nucleic
acids.

Answer 6

polymerase chain reaction

Question 7

PCR advantages

Answer 7

user friendly
more amendable
automated

Question 8

gold standard in the detection of covid 19

Answer 8

rt PCR

Question 9

1983, ___ conceived the idea of amplifying dna in vitro

Answer 9

kary mullis

Question 10

–In the process of working through a
mutation detection method, he
came up on a way to double the test
targe

Answer 10

kary mullis

Question 11

PCR Initially used the “_”
method.

Answer 11

3 graduate student

Question 12

Knew that you could use __ to initiate DNA
synthesis

Answer 12

primers

Question 13

first sample to be used in amplification used by kary mullis

Answer 13

nerve growth factor

Question 14

the first successful sequence to amplify is

Answer 14

short fragment of escherichia coli

pBR322

Question 15

PCR is called previously as

Answer 15

Polymerase Catalyze
Chain Reaction

there’s catalyze because there’s dna polymerase used before

Question 19

REQUIREMENTS for PCR

Answer 19

1. double stranded DNA - template - to give the order of nucleotide
2. Deoxyribonucleotide bases
3. 1 enzyme for addition of nucleotide to growing strands - DNA polymerase
4. primer

Question 20

product of PCR -

Answer 20

amplicons

Question 21

• One copy of dsDNA becoming two copies.
  In vivo
• About an hour or two, PCR can produce millions of
  copies called amplicons of DNA

Answer 21

in vitro PCR

Question 22

• In contrast, it would probably take several days for
  a cell to produce the same number of copies in vivo.

Answer 22

in vivo replication

Question 23

3 graduate steps in pcr

Answer 23

denaturation
annealing
extension

Question 24

temp and time for denaturation

Answer 24

90-96 * C

20-60secs

Question 25

temp and time for annealing

Answer 25

50-70 *C

20-90 secs

Question 26

temp and time for extension

Answer 26

68-75*C

10-60 secs

Question 27

target in pcr

Answer 27

double stranded DNA

Question 28

a process wherein we Reduce double stranded molecules to single stranded molecules

Answer 28

denaturation

Question 29

This is accomplished by heating the samples by 90 or 94-96 *C for 20-60 seconds up to several minutes, depending on the type
of target for testing

Answer 29

denaturation

Question 30

the most critical step in specificity of the pcr

Answer 30

annealing

Question 31

In this step, there are two oligonucleotides, which will prime the
synthesis of DNA and will anneal to the complementary sequence of the
template.

Answer 31

annealing

Question 32

__determines the specificity of the amplification.

Answer 32

Primer

Question 33

The __ dictates the part of the template that would be amplified.

Answer 33

primer

Question 34

the location where will the primer attach during PCR

Answer 34

oligonucleotides

Question 35

what are the 2 types of primers

Answer 35

forward and reverse

Question 36

the type of primer that will hybridize to the complementary strand just below the 5 prime

Answer 36

forward primer

Question 37

the type of primer that hybridizes to the 3 prime of the sequence to be amplified

Answer 37

reverse primer

Question 38

EXTENSION (PRIMER EXTENSION) also called as

Answer 38

elongation

Question 39

This is when the DNA synthesis occurs.

Answer 39

EXTENSION

Question 40

The DNA Polymerase catalyzes the formation of the phosphodiester bonds
in between the incoming DNTPs which is then determined by the hydrogen
bonding to the template.

Answer 40

EXTENSION (PRIMER EXTENSION)

Question 41

bases always attach to which part of the primer

Answer 41

3` end

Question 42

In some cases, the
annealing temperature
is close enough to the
extension temperature
that the reaction can
proceed with only 2
temperature changes.

Answer 42

Two-Step PCR

Question 43

COMPONENTS OF PCR

Answer 43

1. Primers
2. DNA template
3. Deoxyribonucleotide
   Bases
4. DNA Polymerases
5. PCR Buffer
6. Thermal Cyclers
7. Controls for PCR

Question 44

Critical component of PCR because they determine the
specificity of the PCR

Answer 44

primer

Question 45

primer

They are analogous to the _ in blotting and hybridization procedures

Answer 45

probes

Question 46

primers are desifgned using genomic sequence available from

Answer 46

national center for biological information

Question 47

factors that affect primers

Answer 47

1. primer sequence - number of guanine and cytosine - must be a lot
2. length affecting the conditions in which the primer will bind to its target

Question 48

the 2 factors that affecr primer is affected by ___

Answer 48

the melting temperature of the primer

Question 49

served as the starting point in setting optimal annealing - starting point for optimal binding

Answer 49

melting temperature

Question 50

primers (forward) its melting temp can be adjusted in the length of the template and number of guanine and cytosine

t or f

Answer 50

true

Question 51

primers

single stranded DNA fragments that how many bases in length

Answer 51

20-30 bases in length

Question 52

problems in primer

Answer 52

mispriming
primer dimers

Question 53

creation of unintended product caused by primers attaching to wrong area

Answer 53

mispriming

Question 54

how to prevent mispriming

Answer 54

HOT start PRC

Question 55

PCR products that are double the size if the primer, results from the binding of primer with each other (on the 3` end)

Answer 55

primer dimers

Question 56

it connects all bases to each other

Answer 56

phosphodiester bond

Question 57

connects the primer to template

Answer 57

hydrogen bond

Question 58

Clinical sample sources of dna template

Answer 58

all microorganism

Question 59

what kind of sample if we are using hair follicle

Answer 59

mitochondrial DNA

Question 60

PCR reagents and conditions, nanogram amounts of genomic DNA are
sufficient for consistent results

true or false

Answer 60

true

Question 61

how many data template are we using?

Answer 61

❑Routine clinical analysis - 100 ng to 1 μg of
DNA is usually used

Question 62

Lesser amount of data template are required for more
defined template preparations such as
_____ DNA or product from a
previous amplification

Answer 62

cloned target

Question 63

best templates

Answer 63

good condition
free of contaminating proteins
without nicks or breaks that can stop DN synthesis or cause misincorporation of nucleotide bases

Question 64

templates with high GC content and secondary structure may prove more ____ to optimize for amplification

Answer 64

difficult

Question 65

are
the building blocks of DNA

Answer 65

Nucleotide triphosphates

Question 66

Deoxyribonucleotide
triphosphates (dNTPs

Answer 66

1. Adenine
2. Thymine
3. Guanine
4. Cytosin

Question 67

In common PCR applications,
the recommended final
concentration of each dNTP is
generally

Answer 67

0.2 mM