Biology 1.5 PCR and Gel Electrophoresis

Question 1

DNA Analysis

Answer 1

DNA can be extracted from cells, analysed and manipulated by humans. Many techniques are used in these processes, including:

Polymerase Chain Reaction (PCR)
To amplify DNA, producing millions of copies.
Based on semi-conservative replication.

Gel Electrophoresis
To separate fragments of DNA based on size.

Question 2

POLYMERASE CHAIN REACTION (PCR) steps

Answer 2

DNA is denatured by heating to 94℃ - 98℃ (separating strands).

Mixture is cooled (48℃ - 72℃) and primers* anneal (bind) to the DNA, initiating DNA replication using a heat-stable DNA polymerase (Taq).

After primer binding, DNA polymerase extends the synthesis of the DNA (68℃ - 72℃).

After one cycle, the target DNA has produced two copies.

The cycle repeats, doubling DNA each time.

Question 3

Gel electrophoresis

Answer 3

DNA sample is amplified using PCR.

Amplified DNA is cut into fragments using restriction enzymes.

The fragments are then loaded into a gel, submerged in a buffer and an electric current is applied.

DNA is negatively charged and will migrate towards the positive end of the electrophoresis tank.

Larger fragments migrate slowly and don’t move far in the gel.

Smaller fragments migrate quickly and can move further in the gel.

Question 4

APPLICATION: ELECTROPHEROGRAMS

Answer 4

To produce DNA profiles, scientists target short tandem repeats (STRs) via PCR.

These STRs (fragments) vary in length between individuals and their alleles.

The PCR products are separated by gel electrophoresis.

The results of this separation and analysis are presented in figures called electropherograms.

Question 5

APPLICATION: DNA PROFILING

Answer 5

Like a fingerprint, individuals produce a unique pattern of DNA fragments analysed by gel electrophoresis.

The pattern of bands is called a DNA profile.

DNA profiling has a range of uses:

Forensic science

Paternity testing

Identification of people involved in disasters