Brent Cornell

The manipulation of DNA requires a variety of molecular components (enzymes) as well as numerous experimental techniques:

• Enzymes: Polymerase (copies DNA), endonuclease (cuts DNA) and ligase (pastes DNA)
• Techniques: PCR (amplifies DNA molecules) and gel electrophoresis (separates DNA molecules)

Enzymes

Enzymes are a class of proteins that function to catalyse chemical reactions (enabling them to proceed under favourable conditions)

• Polymerase is used to synthesise new strands of DNA from a template molecule (used in PCR to amplify DNA)
• Endonucleases (restriction enzymes) cut DNA backbones to generate blunt ends or complementary sticky ends
• Ligase covalently joins DNA backbones (with phosphodiester bonds) to paste disparate sequences of DNA together
• Reverse transcriptase converts mRNA sequences into cDNA sequences (with no introns) for bacterial transformations

Polymerase Chain Reaction

The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions

• The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample
• Each reaction doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (2³⁰)

The reaction occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:

1. Denaturation – DNA sample is heated (~90ºC) to separate the two strands
2. Annealing – Sample is cooled (~55ºC) to allow primers to anneal (primers designate sequence to be copied)
3. Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC)

Taq polymerase is an enzyme isolated from the thermophilic bacterium Thermus aquaticus

• As this enzyme’s optimal temperature is ~75ºC, it is able to function at the high temperatures used in PCR without denaturing
• Taq polymerase extends the nucleotide chain from the primers – therefore primers are used to select the sequence to be copied

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate and isolate DNA fragments based on their mass / size

• Samples of DNA are placed in the wells of an agarose gel and the gel is then submerged in a buffering solution
• An electric current is applied which causes the samples to move through the gel
• The fragments move towards the positive anode (as DNA is negatively charged due to the presence of phosphate groups)
• Smaller fragments are less impeded by the gel matrix and hence will move faster and further through the agarose gel
• This causes samples of different sizes to separate out as they travel at different speeds

DNA Profiling

DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles

• Within the non-coding regions of the genome there exists satellite DNA – long stretches of DNA made up of repeating elements
• These repeating elements (short tandem repeats – STRs) will vary in length in different individuals (resulting in unique DNA profiles)

Comparative STR Lengths at Two Different Loci

DNA profiling is commonly used in criminal investigations (i.e. forensics) and to settle paternity disputes

The procedure involved is common for both:

• A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR
• STR loci are isolated (either by excising with restriction enzymes or by targeting these regions with specific primers during PCR)
• Fragment lengths will differ between individuals due to the variable length of their short tandem repeats
• The fragments are separated using gel electrophoresis and the resulting profiles are compared

Forensic Investigations:

Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur

The number of loci used to generate a unique profile depends on the size of the population being compared

• E.g. America (population: ~ 320 million) uses 13 loci for comparison; Australia (population: ~ 25 million) uses only 9 loci

Forensic Investigation Example (click on the picture to identify the killer)

Paternity Testing:

Children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments

• In other words, all fragments produced in the child should also be produced by either the mother or father

Paternity Testing Example (click on the picture to identify the father)