Lab 2 EXAM

Name two or more applications and/or ways that PCR is used in science today.

Paternity tests and developing vaccines

What is meant by “exponential” amplification? Approximately how many copies of a targeted sequence of DNA can be made (“amplified”) in 30 cycles?

In PCR DNA amplification proceeds “exponentially” meaning that in each

cycle the number of targeted sequences synthesized doubles in number. After 30

cycles there will be over 1 Billion copies of the target sequence.

1st Step of PCR cycle

Denaturation = approximately 95°C for 1 – 2 minutes; the two strands of

double-stranded DNA separate due to breakdown of hydrogen bonds

2nd step of PCR cycle

Annealing of Primers = approximately 65°C for 30 – 45 seconds; Primers

bind to complimentary strands of DNA on either end of the targeted region

3rd step of PCR cycle

Elongation (aka: extension) = approximately 72°C for 1 1/2 – 2 minutes;

Taq polymerase binds to primers and begins to synthesize a new

complimentary DNA strand along each of the two original single strands

Name the 7 key ingredients that need to be added to each PCR tube/sample

DNA Template, Oligonucleotide Primer 1, Oligonucleotide Primer 2, Taq Polymerase, dNTPs , PCR Buffer, MgCl2

What is the overall goal of PCR? That is, what does PCR do?

PCR is a temperature controlled reaction that synthesizes millions of copies of a

desired segment of DNA. Technically this is referred to as “amplifying” the

targeted DNA sequence.

Elongation

– the stage of PCR when double-stranded DNA is actively being built

(manufactured) from a template strand by Taq Polymerase

Anneal

When two complimentary strands of DNA join via the formation of hydrogen bonds forming between corresponding base pairs

Denature

– The process of DNA strand separation; occurs at very high temperatures (90-95°C)

Taq Polymerase

a specialized heat stable polymerase that synthesizes a

growing chain of DNA nucleotides that are complimentary to each of the

strands of the target sequence. This enzyme builds the new DNA molecules.

Primer

– two short DNA strands (one for each template strand) that flank the

target sequence and provide a site for Taq Polymerase to bind and begin

polymerization

Agarose Gel

a semisolid matrix through which charged molecules that differ

in size (molecular weight) will migrate to varying lengths and therefore allowed

to be sorted

DNA (allele) ladder

a solution containing a mixture of DNA fragments of

known length used to serve as a baseline (molecular ruler) with which to assess

fragments of unknown length.

Buffer

an aqueous solution that is pH balanced, isotonic and provides a neutral environment in which electrophoresis can occur

Anode

the positively charged electrode of the electrophoresis chamber

Cathode

the negatively charged electrode of the electrophoresis chamber

Well

slit-like indentation within the electrophoresis gel into which the DNA sample is loaded prior to electrophoresis