DNA amplification methods

Question 1

In vitro [outside] DNA amplification

Answer 1

Polymerase Chain Reaction (PCR) :

Question 2

In vivo [inside] DNA amplification

Answer 2

Molecular cloning

Question 3

What is PCR

Answer 3

The artificial exponential amplification of DNA targets in a template using DNA polymerases.

Question 4

PCR

DNA pol. I:

2 points

Answer 4

1. Main enzyme for DNA replication in bacteria

2. The DNA polymerases used in PCR belong to this group.

Question 5

PCR Process

4 points

Answer 5

step 1: Double stranded DNA -> Denaturation (95C) -> single stranded DNA

Step 2: Annealing of primers and attachment of DNA polymerase (40C-65C)

Step 3: Extension of primers (68C-72C)

PCR CYCLE DONE

4. cyclic amplification of target sequence -> amplification of target sequence = amplicon

Question 6

Main components of PCR

Answer 6

1. Taq DNA polymerase [stable at high temp]

Question 7

Template for PCR

2 points

Answer 7

1. Double stranded DNA
   Genomic DNA
   Plasmidic DNA
2. Single stranded DNA
   cDNA (reverse transcription of RNA).
   Certain single stranded RNA/DNA viruses.

Question 8

Primers for PCR

Answer 8

1. Short synthetic oligonucleotides with single stranded sequence.
2. 6 nucleotides of length and up.
3. Totally or partially sequence-specific.
4. The 3’ nucleotide must complement the sequence.

Question 9

Temperature cycles in PCR

Answer 9

1. Initial denaturation.
2. Denaturation time is
   dependent on the type of
   template: must be kept to
   the minimum.
3. Annealing temp. and time
   are dependent on the Tm of
   the primer.
4. The extension time is
   dependent on the fragment
   size and on the enzyme used.

Question 10

Thermal cyclers

Two technologies

Answer 10

1. Heat pumps: Peletier effect
2. Heated air: Very rapid cycles
   Circulation of heated air into a chamber containing
   the PCR tubes.

Question 11

Evoloution of thermal cyclers

Answer 11

1. water baths
2. automated heated blocks
3. fast systems
4. real time detection

Question 12

PCR Specificity

Answer 12

1. Primer sequence
2. Template quality and concentration
3. Stringency of the annealing of the primers

Ionic force [Mg+]: high [Mg+] -> Low Stringency

Temperature: high temperature -> High stringency

Question 13

8 PCR applications

Answer 13

1. Gene cloning, discovery and use
2. monitoring gene expression
3. mutagenesis
4. detection of hereditary diseases
5. detection of micro-organisms and viruses
6. detection and quantification of GMOs
7. fingerprinting and polymorphism, phylogeny
8. ancient DNA

Question 14

Whole genome amplification

5 points

Answer 14

1. Amplification of entire genome or entire DNA in a sample.
2. High yield: produces mg of DNA from less than pg of template
3. Phi 29 DNA polymerase

Pyrophosphatase

Exonuclease resistant random primer (binds randomly to DNA)

4. Reactions done at 30 ºC
5. Used to amplify traces of DNA, ancient DNA.

Question 15

In vivo DNA amplification: cloning

Answer 15

1. Consists of amplification of DNA fragments in bacterial hosts:
2. Using plasmid vectors
   Using phagemid vectors
3. Application: cloning of DNA/genes