Module 2.1- Laboratory Techniques (PCR)

Molecular biology

studies the flow of information from DNA to mRNA to protein (DNA replication, DNA transcription to mRNA, mRNA translation to protein)

Molecular cloning

• experimental methods which are used to create recombinant DNA molecules

• essentially a strategy to replicate the natural flow of genetic information on the lab bench

Traditional molecular cloning workflow

Must cut plasmid and gene fragments with the same restriction enzymes (scissors)

• Find DNA sequences

• Amplify (make many copies)

• Assemble (cut and paste)

• Put assembled DNA into host (transformation)

What we need to do practically

Miniprep

Using chemicals to break down the cell membrane

Restriction enzymes

bacterial proteins that cut DNA at specific recognition sites, acting as a defense system against foreign DNA, like viruses

To create compatible sticky ends that allow proper ligation

Why must the plasmid and gene fragment be cut with the same restriction enzyme?

Sticky ends

Overhanging single-stranded DNA sequences that help fragments anneal to each other

Ligation

Process that joins the digested plasmid and gene fragments together

Transformation

Process by which recombinant plasmid is introduced into bacteria

Role of bacterial cells in molecular cloning

Taking up recombinant plasmid and replicating it

A gene insert and a selection marker (e.g., antibiotic resistance gene).

What does the recombinant plasmid contain?

By growing them on selective media (e.g., media containing antibiotics).

How are bacteria carrying recombinant plasmids selected?

Restriction mapping

• laboratory method that checks plasmid size and fragment patterns

• cuts plasmid with enzymes and runs a gel

Sequencing

method that is used to determine the exact DNA sequence of the insert

PCR (Polymerase Chain Reaction)

technique that amplifies DNA for verification

First objective of genetic engineering

to isolate copies of specific genes in pure form

DNA Amplification

Generates multiple copies of a target from a single molecule of DNA or mRNA in vitro

In vitro

outside their normal biological context (e.g., in a test tube in the lab)

a temperature cycle (NOT isothermal)

What does DNA amplification via PCR require?

Isothermal

a process, condition, or system where the temperature remains constant, meaning there is no change in temperature (ΔT = 0)

Isothermal DNA amplification techniques

• rolling circle amplification

• loop mediated amplification

• strand displacement amplification

What happens during each round of amplification?

amount of DNA doubles, leading to an exponential increase in the target DNA

Denaturation

• DNA strands containing the target sequences are separated by heating the reaction mixture to 95°C

• The hydrogen bonds between the two strands break down

Elongation/extension

• Taq polymerase binds to the template DNA and starts adding nucleotides that are complementary to
  the DNA strand

• The solution is heated to 72 °C, as that is the optimum temperature for Taq Polymerase

Annealing

• The process of allowing two sequences of DNA to form hydrogen bonds

• In this step, the primers anneal to the target sequences by cooling the solution to ~55°C

How many times can the PCR cycle be repeated?

24 – 30 times

PCR reagents

• Template DNA

• Sequence-specific DNA primers flanking the target sequence

• Taq polymerase

• Deoxynucleotide triphosphates

• PCR buffer

What are usually the first things to run out?

dNTPs

Template DNA

Contains the target sequences

Taq polymerase

• DNA polymerase from Thermus aquaticus, a bacterium
  found in hot springs

• Able to withstand extremely high temperatures

Primers

Short single strands of RNA or DNA (typically about 18-24 bases)

Distinction between PCR and cellular replication

primers in PCR are DNA

Distinction between PCR and cellular replication

primers in PCR are DNA

Primer design notes

• The length of the primer is important

• Short primers produce inaccurate, nonspecific DNA amplification product

• Long primers result in a slower hybridizing rate

Primer properties

1. Length of 18– 24 bases

2. Melting temperature (Tm) of 50-60°C

3. 40-60% G/C content

4. Start and end with 1-2 G/C pairs (strong binding to the ends are important –
   especially at the 3’-end)

5. Primer pairs should have a Tm of within 5°C of each other

6. Primer pairs should not have complementary regions

7. Try to avoid long stretches of di-nucleotides (e.g. ATATATATATATA)

Melting temperature (Tm)

• 50-60 degrees C for efficient annealing

• Primers with a higher Tm than 65 tend to result in secondary annealing

• Tm = 2 degrees C x (A+T) + 4 degrees C (G+C)

Annealing temperature (Ta)

• Ta of primers should be within 5 degrees C of each other

  • If they vary greater than 2°C to 5°C from each other, the primer with the higher Tm will have a higher chance of mispriming at the lower temperature. The primer with the lower Tm might not be functional at a very high temperature

  • Ta = Tm – 5 °C

What is Ta dependent on?

• A/C/T/G composition

• pH of the reaction, salts present, etc.

Why is the 3’ end of a primer is critical (last 5 – 6 nucleotides) in the success of a
PCR?

• DNA polymerase can only add nucleotides to a free 3’ hydroxyl (on the primer)

• Therefore, to design primers, you must start at the 3’ end of the template
  DNA

• Perfect base pairing between the 3’ end of the primer and the target DNA
  template is important

• Additionally, including G’s and C’s in this section will increase binding to
  the template

Primers

• Short single strands of RNA or DNA (typically about 18- 24 bases)

• Serves as a starting point for DNA synthesis:

  • DNA polymerases can only add new nucleotides to an existing strand of DNA

• Researchers custom design primers to be complementary to the beginning and end of their target DNA sequence (forward and reverse primers)

• Both complementary strands amplified simultaneously

Distinction between PCR and cellular replication

Primers in PCR are DNA

Issues with primers

• Primers that match multiple sequences will give multiple products

• Repeated sequences can be amplified – but only if unique flanking regions can be found where primers can bind

• A primer may form a dimer with itself of with the other primer’

• Primers can also have self-annealing regions within itself (i.e. hairpin and foldback loops)

Dimer

A molecule or complex formed when two individual units (monomers) join together

How can DNA be quantified?

By measuring the absorbance at 260 nm using a spectrophotometer 

• Nucleic acids absorb UV light at 260 nm —> aromatic moieties in purines (thymine, cytosine, and uracil) and pyrimidines (adenine and guanine) structure

Calculating amount of template DNA (N0)

1 A260 = approximately 50 micrograms/mL

Number of target DNA sequences at the end of the PCR cycle

N = 2^t (N0)

• N = number of copies after amplification

• t = number of cycles

• N0 = number of copies initially present in the reaction

Duration and temp for each step

1. Denaturation = 15-30 sec at 95 degrees C

2. Annealing = 30 sec at 5 degrees C

3. Extension = 72 degrees C (time not specified)

DNA polymerase and amplicon length

What are extension times dependent on?

Temperature of extension time

68-72 degrees C

General rule when using Taq polymerase

Use extension times of 1 min per 1000 base pairs

• For products less than 1 kb, use 45-60 seconds

• Products greater than 3 kb or reactions using more than 30 cycles may require longer extensions 

Taq polymerase

A heat-stable DNA polymerase from the bacterium Thermus aquaticus that is essential for PCR because it can withstand the high temperatures required for DNA denaturation

Typical PCR protocol for 500 base pare amplicon

• 1 cycle at 95 degrees C for 2 min 

• 25 cycles at 95 degrees C for 15 sec (denaturation), 55 degrees C for 30 sec (annealing), 72 degrees C for 45 sec (extension)

• 1 cycle at 72 degrees C for 5 min (to finish replication on all templates)

• 1 cycle at 4-10 degrees C for an indefinite period (storing the sample prior to further analysis) 

Amplicon

A piece of DNA or RNA that is the source or product of a replication or amplification event

Negative controls for a PCR reaction

1. No template control (NTC)

2. No amplification control (NAC)

No template control (NTC)

Omits any DNA template from a reaction —> a general control for extraneous nucleic acid contamination

No amplification control (NAC)

Omits the DNA polymerase from the PCR reaction —> control for bakground signal that is not a function of the PCR

Applications of PCR

• Genetic Research (i.e. mapping techniques in the Human Genome Project relied on PCR)

• DNA fingerprinting

• Detection of bacteria or viruses (i.e. HIV)

• Diagnosis of genetic disorders

Amplifying DNA

• PCR can be used amplify linear DNA or portions of DNA from a plasmid

• If you want to amplify plasmid DNA (i.e. the whole plasmid) we can transform the plasmid into a bacteria, grow it, and then extract the DNA