Lab In Microbiology 3: Determination Of Insert Orientation By The Polymerase Chain Reaction

Front 1

What is PCR?

Back 1

An in vitro method for amplifying selected nucleic acid sequences

Front 2

What does the PCR method consist of?

Back 2

Repetitive cycles of DNA denaturation, annealing and extension by heat stable DNA polymerases

Front 3

What type of primers are used for PCR?

Back 3

Synthetic oligonucleotides

Front 4

What are requirements for the synthetic oligonucleotides?

Back 4

-They must have different sequences
-THey must be complementary to sequences on OPPOSITE strands of the template DNA
-They must flank the segment of DNA that is to be amplified

Front 5

What is the first step of PCR?

Back 5

THe template DNA is denatured by heating in the presence of a large molar excesss of each of the two primers and the four dNTPs.

Front 6

What is the second step of PCR?

Back 6

The reaction is cooled to a temperature that allows the primers to anneal to their single stranded complementary target sequences.

Front 7

What is the third step of PCR?

Back 7

DNA polymerase extends the annealed primers.

Front 8

What type of reaction is PCR and why?

Back 8

Exponential. At the end of each cycle the amount of DNA doubles.

Front 9

What are the termini of the PCR end product defined by?

Back 9

The 5' termini of the oligonucleotide primers.

Front 10

What is the length of the PCR end product defined by?

Back 10

The distance between the primers

Front 11

What is often added to PCR primers to aid handling and insertion of amplified DNA into a vector?

Back 11

Endonuclease target sequences (cut sites)

Front 12

What was the original polymerase used to catalyse the extension of annealed primers in PCR?

Back 12

The Klenow fragment of E. coli DNA polymerase 1

Front 13

What was the problem with using the Klenow fragment for PCR?

Back 13

This enzyme is denatured and inactivated by the high temperature requied for the denaturation of DNA. So each round of PCR requires the addition of new enzyme.

Front 14

How was this problem solved?

Back 14

A thermostable DNA polymerase was purified.

Front 15

From which organism was the thermostable DNA polymerase purified and what was the enzyme called?

Back 15

Thermus aquaticus, and the enzyme was called Taq polymerase.

Front 16

What are some characteristics of the bacterium and the enzyme?

Back 16

Thermus aquaticus is a thermophilic bacterium. The enzyme can survive extended incubations at 95 degrees or repeated cycles (30-50) of denaturation during PCR.

Front 17

When is Taq polymerase most active and what problem does this solve?

Back 17

It is most active at 72-74 degrees. This solves the problem of mispriming which often occurs when primer extension is performed at low temperatures, such as the 37 degrees normally required for elongation with regular DNA polymerases.

Front 18

In a PCR reaction, what is the duration of the elongation period determined by?

Back 18

The distance separating the two oligonucleotide primers.

Front 19

What is the rate of DNA synthesis by most heat stable DNA polymerases?

Back 19

750-1000 base pairs per minute

Front 20

How long would elongation take for the 350 bp amplimer seen in this exercise?

Back 20

Less than 30 seconds

Front 21

What are some additional characteristics of new thermostable DNA polymerases?

Back 21

They have improved thermoresistance and proof reading activitiy.

Front 22

What direction is DNA polymerase proofreading activity?

Back 22

3' to 5'

Front 23

What is one of the most widely used DNA polymerase and wha torganism does it originate from?

Back 23

Vent DNA polymerase, which originates from Thermococcus litoralis

Front 24

What is the half-life of Vent and how does this compare to Taq?

Back 24

Vent has a half life of 6.7 hours at 95 degrees while Taq has a half life of 1.2 hours.

Front 25

Is Vent or Taq more accurate?

Back 25

Vent is about 6 times more accurate than Taq.

Front 26

Which procedures require proofreading activity and which ones do not necessarily?

Back 26

Cloning and sequencing require proofreading, while simple diagnostic procedures do not need such accuracy.

Front 27

What type of accuracy does the use of PCR to introduce random mutations require?

Back 27

The least accurate enzyme

Front 28

What is site directed mutagenisis and what type of accuracy in enzyme acitvity does it require?

Back 28

A unique mutation is introduced at a precise location and therefore requires the most accurate enzyme

Front 29

At what temperatures does denaturation occur?

Back 29

95 degrees

Front 30

At what temperatures does primer annealing occur?

Back 30

55 degrees

Front 31

How long is the first hold, at what temperature and what does it do?

Back 31

The first hold is at 95 degrees for twelve minutes. It ensures that the two template strands are separated

Front 32

At what temperature does elongation occur?

Back 32

72 degrees

Front 33

At which cycle is the desired PCR product achieved?

Back 33

The third cycle

Front 34

What is rescue PCR?

Back 34

Performing PCR directly on colonies obtained after transformation and cloning. THis allows the rapid screening of colonies without having to make plasmid preparations of each clone.

Front 35

If one uses primers specific for the vector, flanking the insert, what would a small band appearing for no insert represent?

Back 35

A small primer dimer

Front 36

What type of primers are required to ensure that the orientation of the insert is correct?

Back 36

This is known as DIRECTIONAL PCR. One oligonucleotide is specific for the vector, and a second is specific for the insert.

Front 37

What is directional cloning?

Back 37

Two different restriction sites are used to introduce a cDNA into a vector in a specific oritenation.

Front 38

What type of primers are used in THIS exercise?

Back 38

A 5' (sense) oligonucleotide derived from the sequence of the GST encoding plasmid a few nt upstream from the BamH1 site.
A 3' (antisense) oligonucleotide from the sequence at the 3' region of the first SH2 domain (nt 306 of zap-70)

Front 39

What is the positive control in this exercise?

Back 39

A plasmid known to contain the insert in the correct orientation

Front 40

What is the negative control in this exercise?

Back 40

The empty pGEX-3T vector

Front 41

What are the faint bands ABOVE the 350 bp amplimer?

Back 41

Non-specific bands due to mispriming

Front 42

What is the hzy band at the bottom created by?

Back 42

Non-specific dimerization of primers

Front 43

In a Southern hybridization assay, using a specfic oligonucleotide sequence within the zap-70 amplimer was used, which bands would show hybridization?

Back 43

Only the 350 bp bands

Front 44

Why is mineral oil used in this exercise?

Back 44

To prevent the evaporation of the samples during repeated denaturation steps of the PCR reaciton.

Front 45

What type of organism likes to live at 9 degrees?

Back 45

Psychrophiles

Front 46

What type of habitat sizewise do psychophiles live?

Back 46

They have a large habitat (90% of the ocean is 5 degrees or colder)

Front 47

What is an example of a psychrophile?

Back 47

Chlamydomonas nivalis (pink spores)

Front 48

What organism likes to live at 24 degrees?

Back 48

Psychotroph (or faculatative psychrophiles)

Front 49

What form do we most often find psychotrophs?

Back 49

In spoilage of refrigerated food

Front 50

What is the name for most human pathogens and at what temperature do they like to grow?

Back 50

Mesophiles, like to grow at 37 degrees.

Front 51

What type of organism likes to grow at 68 degrees?

Back 51

Thermophiles

Front 52

What domain do most thermophiles belong to?

Back 52

Most thermophiles are procaryotes

Front 53

Where are thermophiles found?

Back 53

Composts, hot water lines, hot springs

Front 54

What type of organism likes to grow at 95 degrees?

Back 54

Hyperthermophiles

Front 55

What are some examples of hyperthermophiles?

Back 55

Thermococcus litoralis, Thermus aquaticus