PCR and primer design

Question 1

What is PCR?

Answer 1

A method for selectively amplifying a particular segment of DNA

Question 2

What can PCR be thought of as?

Answer 2

A molecular photocopier

Question 3

What can we do with a PCR product?

Answer 3

Digest with restriction enzymes
Sequence
Clone

Question 4

Who invented PCR?

Answer 4

Kary Mullis

Question 5

When was PCR invented?

Answer 5

In 1983

Question 6

When was the first published account of PCR?

Answer 6

In 1985

Question 7

In what year was the work on PCR awarded the Nobel Prize for Chemistry?

Answer 7

In 1993

Question 8

How long should PCR primers be?

Answer 8

~20 bases long

Question 9

What should the GC content of PCR primers be?

Answer 9

45-55%

Question 10

What should the 3’-most base of a PCR primer ideally be?

Answer 10

G or C

Question 11

What might a segment of DNA amplified by PCR represent?

Answer 11

A small part of a large and complex mixture of DNAs, e.g. a specific exon of a human gene

Question 12

PCR is a photocopier capable of duplicating a part of a sentence or paragraph. What must there be locations for and what must be special about these locations?

Answer 12

Must be locations for the beginning and the end of the section to be copied that are identifiable
Locations must be unique to allow the copier to locate the correct piece of text

Question 13

How long does it take PCR to amplify a usable amount of DNA (visible by gel electrophoresis)?

Answer 13

~2 hours

Question 14

Template DNA for PCR needs to be highly purified, true or false?

Answer 14

False, can use a colony of bacteria or yeast

Question 15

What is PCR powerful enough to amplify?

Answer 15

A single DNA molecule, e.g. from a single sperm cell

Question 16

Prior to PCR, what did Southern blotting (1975) permit?

Answer 16

The rudimentary mapping of genes in unrelated individuals (RFLPs, insertions and deletions)

Question 17

Prior to PCR, what did DNA sequencing (1978) require?

Answer 17

Genes to first be cloned into bacteria using vectors such as plasmids of lamda phage

Question 18

Prior to PCR, what were the downsides to gene library construction and screening?

Answer 18

Could take many months and libraries had to be made for each individual genome analysed

Question 19

Prior to Mullis, who invented the basic principle of replicating a piece of DNA using two primers in 1971?

Answer 19

Gobind Khorana

Question 20

What was Khorana’s principle of PCR limited by and how did Mullis improve on it?

Answer 20

Progress was limited by primer synthesis and polymerase purification issues
Mullis properly exploited amplification

Question 21

How many cycles are there in a basic PCR reaction?

Answer 21

30-35 cycles

Question 22

What steps do the PCR cycles comprise of?

Answer 22

Denaturation, 95 degrees C, 30 seconds
Annealing, 55-60 degrees C, 30 seconds
Extension, 72 degrees C, time depends on product size

Question 23

What is needed for a basic PCR reaction mix?

Answer 23

Template DNA
Reaction buffer
Nucleotides (dNTPs)
Primers
DNA polymerase

Question 24

What does a PCR reaction buffer typically contain?

Answer 24

Tris
Ammonium ions
Potassium ions
Magnesium ions
Bovine serum albumin
Detergents

Question 25

What usually is the DNA polymerase used for PCR reactions?

Answer 25

Taq polymerase

Question 26

At which cycle do target products begin to get made?

Answer 26

The third cycle

Question 27

How many copes of DNA are there at 30 cycles?

Answer 27

~E9 target copies | 60 other DNA copies whose length is undefined

Question 28

The accumulation of target products in PCR is not strictly a doubling at each cycle in the early phase, true or false?

Answer 28

True

Question 29

Increasing the cycle number above ~35 increases the amount of target product generated, true or false?

Answer 29

False, increasing the cycle number above ~35 has little positive effect

Question 30

When does there become a plateau of target products?

Answer 30

When... The reagents are depleted The products re-anneal The polymerase becomes damaged

Question 31

What causes the accumulation of unwanted products in PCR?

Answer 31

When too many cycles are performed

Question 32

Prior to thermal cyclers, how was cycling initially performed?

Answer 32

Using three water baths: one at each cycle temperature

Question 33

When were thermal cyclers introduced?

Answer 33

1986

Question 34

Why did early polymerases have to be replenished at each cycle?

Answer 34

Because they were not thermostable

Question 35

What does Taq stand for?

Answer 35

Thermus aquaticus

Question 36

When was Taq DNA polymerase first described?

Answer 36

1988

Question 37

What does the 37 degree C temperature cause that results in unwanted products?

Answer 37

Non-specific priming

Question 38

Describe PCR cyclers

Answer 38

Available from many suppliers Many block formats & multi-block systems Reactions in tubes, tube-strips, 96-well & 384-well microtitre plates

Question 39

What happened to PCR and the use of Taq polymerase in PCR in the early days?

Answer 39

They were both patented

Question 40

When did PCR patents expire in Europe?

Answer 40

On 28th March 2006

Question 41

In the early days, fees were levied on enzyme and thermal cycler purchases, even for academic use, true or false?

Answer 41

True

Question 42

When was the original Taq patent obatined by Cetus (now owned Roche) ruled invalid?

Answer 42

1st April 2003

Question 43

What part of PCR may still be patented?

Answer 43

Engineered polymerases

Question 44

What is the next step if PCR has not worked?

Answer 44

May need to optimise the reaction conditions

Question 45

How can you check a sample using gel electrophoresis?

Answer 45

Is the product of correct size? How many bands are present? Is any product visible at all?

Question 46

Outline the ways in which the PCR reaction can be optimised

Answer 46

Annealing temperature of the primers Concentration of Mg2+ in the reaction Reduce the amount of template DNA and polymerase - 'less is more' The extension temperature (A/T rich region?) The extension time The denaturing and annealing times

Question 47

What equipment can you use to optimise the annealing temperature?

Answer 47

A PCR cycler with a gradient function

Question 48

How can you optimise the annealing temperature?

Answer 48

In temperature steps of 2 degrees C above and below

Question 49

Primers have a calculated annealing temperature, but how must temperature be confirmed in order to optimise it?

Answer 49

Practically

Question 50

When optimising the Mg2+ concentration in a PCR reaction, what can it sometimes be a compromise between?

Answer 50

Yield and specificity

Question 51

What does the fidelity of PCR depend on?

Answer 51

Mg2+

Question 52

When optimising the Mg2+ concentration, what is Mg2+ varied in steps of?

Answer 52

0.5mM

Question 53

How much template DNA is sufficient for amplification of human genes?

Answer 53

Around 10ng

Question 54

What should the amount of template DNA be reduced or increased to suit?

Answer 54

Genome complexity

Question 55

If you are re-amplifying DNA, what should you do to the DNA first?

Answer 55

Dilute it extensively

Question 56

What should you follow for the amount of enzyme?

Answer 56

The manufacturer's recommendations

Question 57

If using concentrated enzyme, what should you do before setting up the PCR reactions?

Answer 57

Make a 'master mix'

Question 58

What shouldn't you do when adding enzyme to a PCR reaction mix?

Answer 58

Adding more 'just to be certain'

Question 59

Most PCR protocols recommend a long initial denaturation step of 5-10 minutes although this may not always be necessary. When will the longer step be needed?

Answer 59

If... Using a hot-start enzyme that must be activated The starting template is a bacterial cell suspension The target DNA lies within a GC-rich region of DNA

Question 60

Why do some PCR protocols recommend a 30 minutefinal extension step?

Answer 60

Because non-templated A base addition can leave the 3' ends 'ragged'

Question 61

What is the disadvantage to 3' ragged ends in a PCR product?

Answer 61

Causes 'double' peaks when analysing PCR product length by capillary electrophoresis

Question 62

Addition of the non-templated A is very inefficient, hence the need to allow lots of time in the final extension, true or false?

Answer 62

True

Question 63

What does Taq DNA polymerase lack that is commonly present in some other polymerases, e.g. E. coli DNA polymerase I?

Answer 63

The 3' to 5' proof-reading activity

Question 64

How often does Taq-misincorporate a base?

Answer 64

1 base in E4

Question 65

What proportion of a 400 base pair target will contain errors after 20 cycles? Will the error distribution be evenly distributed or random?

Answer 65

33% of molecules | Error distribution will be random

Question 66

Do errors in a PCR product matter? Why?

Answer 66

Yes if you want to clone the amplified DNA An individual molecule may harbour several mutations No, if you want to sequence the amplified DNA or cut it with restriction enzymes

Question 67

How can you overcome errors in PCR products?

Answer 67

Use a proof-reading thermo-stable enzyme, such as Pfu, rather than Taq, or a mix of Pfu and Taq polymerases

Question 68

What size range are amplification products typically?

Answer 68

100-1500 base pairs

Question 69

What is the size of the PCR target limited by?

Answer 69

The integrity of the starting target DNA (less than 50kb)

Question 70

Longer PCR targets are amplifiable (larger than 25kb), but what does it require?

Answer 70

Modified reaction buffer Cocktails of polymerases Longer extension times

Question 71

Can RNA be amplified?

Answer 71

Not directly | The DNA polymerase requires DNA template and will not copy RNA

Question 72

How can RNA be amplified?

Answer 72

mRNA can first be copied into cDNA using reverse transcriptase

Question 73

cDNA is a template for PCR and it need not be double-stranded, true or false?

Answer 73

True

Question 74

PCR products ought to ligate easily into a blunt-ended restriction enzyme site but there is a lower than expected efficiency. Why?

Answer 74

PCR products are not truly blunt-ended as Taq polymerase often adds a single non-templates base (usually A) to the 3' end

Question 75

How can the addition of non-templated bases 3' of the PCR product be taken advantage of?

Answer 75

For TA cloning Linearise the vector at a blunt-ended restriction enzyme site (e.g. EcoRV or SmaI) Incubate the linear vector with Taq polymerase and dTTP to add non-templated Ts Ligate together

Question 76

When designing PCR primers, within how many degrees C should individual annealing temperatures be of one another?

Answer 76

Within 1 degrees C

Question 77

When designing PCR primers, what must primers not base pair with?

Answer 77

Each other With themselves Form hairpins

Question 78

When designing PCR primers, what must primers not anneal to?

Answer 78

Repetitive DNA elements, e.g. Alu elements in the human genome

Question 79

Give an example of how a badly designed primer might form a hairpin

Answer 79

Self-complementary and be able to fold into a hairpin | The ' end of the primer is base-paired, preventing it annealing to the target DNA

Question 80

Give an example of how a badly designed primer might form a dimer

Answer 80

Forms a dimer with itself or with the other primer

Question 81

Primer dimers can be an excellent but unwanted substrate for the Taq polymerase, true or false?

Answer 81

True

Question 82

It is widely recognised that the manual design of PCR primers is difficult and unreliable, so what have been devised which takes all of the design criteria into account?

Answer 82

Computer programs | Some are commercial, others free and open

Question 83

What program at the Whitehead Institute for designing primers is probably the most reliable and versatile free tool currently available?

Answer 83

Primer3

Question 84

What does Primer3 design primers on the basis of?

Answer 84

The DNA sequence entered

Question 85

Why do we need to consider the whole genome and not just the segment of interest when designing PCR primers?

Answer 85

Because the primers might also amplify other segments whose sequence has not been taken into account

Question 86

What does UCSC In-Silico PCR search?

Answer 86

The entire human genome looking for potential primer sites within 4000 bases of one another

Question 87

What information does UCSC In-Silico PCR provide?

Answer 87

It performs a virtual PCR reaction if it finds other potential primer sites Identifies the amplified region, its size and the primer melting temperatures Provides a link to the genomic location

Question 88

Are primer design tools linked to sequence variant databases?

Answer 88

No

Question 89

What factors are there that might mean that only one allele of a DNA fragment gets amplified using PCR?

Answer 89

If a SNP occurs near the 3' end of a primer | If a long length polymorphism (e.g. a LINE1 insertion) occurs within the target region

Question 90

Primer design is based on reference DNA sequences but what else should be considered?

Answer 90

Variants

Question 91

What program can be used to check SNPs and other variants of a DNA sequence?

Answer 91

SNPCheck3

Question 92

What does SNPCheck3 do?

Answer 92

Locates the primers in the genome Performs a virtual PCR Identified SNPs and variants under the primers Identified SNPs and variants in the amplified region