POLYMERASE CHAIN REACTION

Question 1

THE HUMAN GENOME PROJECT
Started in: ___
Goal: ___

Answer 1

Started in 1990
Goal: Fully sequence the entire
human genome

Question 2

– the genome is LARGE

Answer 2

SPECIFICITY

Question 3

– amount of DNA samples are VERY small

Answer 3

AMPLIFICATION

Question 4

Came upon a way to double his test target DNA by giving him 21 copies

Repeating N times would yield: 2N

Answer 4

Dr. Kary Mullis

Question 5

Double-stranded DNA is separated into 2 single strands

what PCR step?

Answer 5

Denaturation

Question 6

Denaturation
Temperature:

Answer 6

94-96oC

Question 7

MOST CRITICAL step for specificity
* Primers hybridize to their
complementary DNA sequences

Answer 7

Annealing

Question 8

Annealing
Temperature:

Answer 8

50-70oC

Question 9

Melting Temperature (Tm) formula?

Answer 9

𝑇𝑚 = 81.5 deg 𝐶 + 0.41(%𝐺𝐶) − (675/𝑛)

Question 10

Temperature at which ½ of the double-stranded DNA exist as single-stranded DNA

Affected by:

Answer 10

(1) Reaction conditions, (2) Salt concentration, (3) Mismatches,
(4) Template condition, (5) Secondary structure

Question 11

Performed by DNA polymerase

Answer 11

Extension

Question 12

Extension

• Catalyzes formation of the
  ___ between dNTPs and the 3’ end of the primer
• Temperature: ___

Answer 12

phosphodiester bonds (1)
68-72oC (2)

Question 13

• Single-stranded DNA fragments
• 20-30 bp long

Answer 13

PRIMERS

Question 14

• 3’ to the sequences amplified
• Hybridizes with the plus strand

Answer 14

REVERSE PRIMER

Question 15

5’ to the sequences amplified
* Hybridizes with the minus strand

Answer 15

FORWARD PRIMER

Question 16

T OR F?
Melting temp (Tm) of the forward and reverse primer must be different

Answer 16

F : MUST BE SIMILAR

Question 17

NA Template
Routine clinical analyses requires:

Answer 17

100 ng – 1 μg

Question 18

Building blocks of DNA

Answer 18

DEOXYRIBONUCLEOTIDE BASES

Question 19

• Thermus aquaticus
• Thermostable
• Good fidelity

Answer 19

Taq polymerase

Question 20

• Thermus thermophilus
• Also has reverse transcriptase
  activity
• Used in RT-PCR (RNA template)

Answer 20

Tth polymerase

Question 21

Provide optimal conditions for enzyme activity

Answer 21

PCR BUFFER

Question 22

Provides pH for optimal
enzyme activity and accurate
amplification

What am i and what pH???

Answer 22

Tris buffer (1)
pH 8 – 9.5 (2)

Question 23

Affect denaturing and
annealing temperatures

Answer 23

Monovalent CATIONS
(KCl and (NH4 )2SO4)

Question 24

T OR F?
↑ salt concentration = shorter
DNA sequences denature
slower

Answer 24

F : longer

Question 25

(MgCl2) what cations?

Answer 25

divalent CATIONS

Question 26

Rapidly & automatically change to the required temperatures for each step and holds it there for designated periods

Answer 26

Machine: thermal cycler

Question 27

* Negative control without DNA * “Contamination” control

Answer 27

Reagent Blank

Question 28

* Negative control with lacking the target sequence

Answer 28

Negative template

Question 29

✓ Enzyme is active ✓ Buffer is optimal ✓ Primers are priming ✓ Machine is working

Answer 29

Positive

Question 30

Major cause of contamination:

Answer 30

presence of PCR products from previous amplifications

Question 31

Induces base damage that catalyzes the formation of single- and double-stranded breaks in DNA

Answer 31

PHYSICal: UV Light

Question 32

PHYSICal: UV Light Enhanced effectivity with the addition of ___

Answer 32

psoralens

Question 33

Widely used method for decontamination and workspace prep

Answer 33

CHEMICAL: 10% Bleach

Question 34

Substitutes the dTTP for dUTP in the PCR reagent master mix

Answer 34

Chemical: dUTP-UNG system

Question 35

Chemical: dUTP-UNG system ___ degrades any nucleic acid containing uracil

Answer 35

Uracil-N-glycosylase (UNG)

Question 36

* Carries the primer sequence and becomes a target for the next amplifications

Answer 36

MISPRIMES

Question 37

* Primers binding to each other

Answer 37

PRIMER DIMERS

Question 38

T OR F??? Primers can bind sequences other than their exact complements on the target (low stringency)

Answer 38

T

Question 39

* Resolving amplification products * Agarose digestion with β-agarase or with iodine incubation

Answer 39

AGAROSE GEL ELECTROPHORESIS

Question 40

✓ Using spin columns ✓ Shrimp alkaline phosphatase (SAP) + exonuclease I (ExoI)

Answer 40

Residual component removal

Question 41

More than one primer pair added to PCR that are primed simultaneously

Answer 41

Multiplex PCR

Question 42

More than one primer pair added to PCR that are primed simultaneously

Answer 42

Nested PCR

Question 43

Starting material is RNA and converted to DNA by reverse transcriptase Used in gene expression studies

Answer 43

Reverse transcriptase pcr “RT-PCR”

Question 44

Detects how much of the target sequence is present usually by fluorescence

Answer 44

REAL-TIME PCR