Ex. 26: DNA Amplification of Mycobacterium tuberculosis by

Ex. 26: DNA Amplification of Mycobacterium tuberculosis by

PCR is DNA replication in a test tube

Objectives ??

Invented by Kary Mullis, 1983. Nobel Prize 1993.

“I was working for Cetus, making oligonucleotides. They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. I was driving with Jennifer Barnett to a cabin I had been building in northern California. She and I had worked and lived together for two years. She was an inspiration to me during that time as only a woman with brains, in the bloom of her womanhood, can be. That morning she had no idea what had just happened. I had an inkling. It was the first day of the rest of my life.”

- from Karry Mullis’s autobiography at the Nobel e-Museum

Did He Really Invent PCR?The basic principle of replicating a piece of

DNA using two primers had already been described by Gobind Khorana in 1971:– Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.

Progress was limited by primer synthesis and polymerase purification issues.

Mullis properly exploited amplification.

What is the Polymerase Chain Reaction?

• Think of it as a molecular photocopier

• It is a means of selectively amplifying a particular segment of DNA.

• The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene.

A Molecular PhotocopierA photocopier capable of duplicating a part of a

sentence:

“The next day was quite a different day. Instead of being hot and sunny, it was cool and misty. Pooh didn’t mind for himself, but when he thought of all the honey the bees wouldn’t be making, a cold misty day always made him feel sorry for them.” A.A. Milne, 1928.

The words in blue must be unique for the copier to locate the correct piece of text.

How Powerful is PCR?PCR can amplify a single DNA molecule, e.g.

from a single sperm.

PCR can amplify DNA to a usable amount (visible by gel electrophoresis) in ~2 hours.

The template DNA need not be highly purified — a boiled bacterial colony.

The PCR product can be digested with restriction enzymes, sequenced or cloned.

The Basics of PCR Cycling 30 - 35 cycles, each comprising:

– Denaturation (95°C), 30 sec.

– Annealing (55–60°C), 30 sec.

– Extension (72°C), time depends on product size.

DolanDNA Learning Center:

PCR Tutorial

DolanDNA Learning Center

on YouTube

Additional narrated PCR Tutorial

What’s in the Reaction?

1. Template DNA2. Reaction buffer and magnesium

ions3. Nucleotides (dNTPs)4. Primers5. DNA polymerase (usually Taq)

So Then, it’s Easy?

Cycling performed with three water baths.

Thermal cyclers introduced in 1986.

Early polymerases were not thermostable, so had to be replenished each cycle.

The 37°C temperature caused non-specific priming, resulting in unwanted products.

Taq (Thermus aquaticus) DNA polymerase first described in 1988.

Forensic Microbiology• Primer for a specific organism will

allow for detection that particular organism

• Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle

• Reverse-transcription PCR (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA

RT-PCR with a norovirus primer

Textbook Clinical Focus, p. 266

Materials needed per 2 tables:Four 200 µl PCR tubes containing PCR beadsMicropipette and tips capable of measuring out 10 µlPatient samples Positive control sampleDistilled water for negative controlEquipment: Nano centrifuge, PCR Thermocycler

Work in a team of 8 students.

Day 1

Materials needed per table: Amplified PCR products from

last session Bottle of 0.5 X TBE (Tris-

borate/EDTA) buffer 50 ml measuring cylinder,

250ml Erlenmeyer flask Minigel electrophoresis

apparatus with gel tray, spacers, and comb

micropipettes and matching tips Microcentrifuge tube containing

20 µl of 10x gel loading buffer.

Materials shared by whole class:1. Scales, flasks, electrophoresis

grade agarose, cylinder, and TBE buffer at “gel making station”

2. Microwave 3. Power source (4 available per

class)4. Nanofuge (2 per room)5. DNA standard (instructor handles

it)6. “InstaStain” ethidium bromide

staining sheets (handled or supervised by the instructor).

7. Gel documentation system

Part 2 of Day 1Cast, Load, and Electrophorese a 1.5 % Agarose Gel

Understanding Gel Electrophoresis

Make 30 ml of a 1.5% agarose gel. How?

How does gel electrophoresis separate DNA fragments?

• Gel acts as sieve to filter DNA fragments• DNA fragments are naturally negatively charged

(phosphate backbone)• DNA pulled towards anode (pos. electrode) by

electric attraction• Smaller fragments move faster through the gel

and larger fragments move slower• gel electrophoresis is optimized by adjusting

agarose concentration in gel

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + DNA is negatively charged and therefore repelled from the negative pole and attracted towards the positive pole

_ _ _ _ _ _ _ _ _ _ _ _

+ + + + + + + + + + + + +

A Typical Image of an Agarose Gel Under UV Light

Decreasing DNASize

Largest DNA fragments

Smallest DNA fragments

The Intensity of the Band is Proportional to the Concentration Of DNA

• An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band

The upper band has far lessDNA when compared to the lowerband. The intensity of the bands are proportional to the amount of DNA at that position in the gel

Sizing The Fragments of DNA

The sizes of the various fragments can be identified by including a “ladder” in the gel– A ladder is a mixture of DNA fragments of known

size – A ladder is usually run beside the unknown

sample so that the sizes of the various DNA fragments in the sample can be identified

Marker / Ladder / Size Standard

• Mixture of DNA fragments of selected sizes

• When run in a gel, fragments will separate into distinct bands that can be used as references

• Fragment size always stated as [X] base pairs (bp)

• Two common ladders: 200bp and 1kbp (1000 bp) ladders

• 200 bp ladder composed of a mixture of small fragments (200 to 4000 bp)

• Ladders commercially available

Sizing a Gel Product

BasePairs(bp)40003000200016001000

500

1Kbp Sample ladder

2000 bp

1000 bp

40003000

20001600

1000

500

Sample 1Kbp ladder

Size of this Fragment?

100

200

400300

600

12001500

1000

500

Size In Base Pair (bp)

The size of the fragment is…??

40003000

20001600

1000

500

?

1 kbp ladder was used

Music video from "Scientists for Better PCR" and Bio-Rad.