ways of manipulating DNA, including the use of polymerase chain reaction (PCR) to amplify DNA and gel electrophoresis to separate DNA fragments, with reference to DNA profiling

What this dot point is asking

VCAA wants the techniques for manipulating DNA in the lab: amplifying it with PCR, separating fragments with gel electrophoresis, and how they combine for DNA profiling (paternity, forensic identification, disease diagnosis).

The answer

Polymerase chain reaction (PCR)

PCR is a technique that amplifies a specific stretch of DNA from tiny starting amounts to millions of copies, fast enough to be useful in diagnostic, forensic and research settings. Invented by Kary Mullis (Nobel 1993).

Ingredients in the reaction tube:

• Template DNA containing the target sequence (can be as little as one molecule).
• Two primers: short (about 20 nucleotide) single-stranded DNA pieces that bind to the flanking sequences on either side of the target. One primer matches each strand. The primers define the region that will be amplified.
• Free deoxynucleotide triphosphates (dNTPs): dATP, dTTP, dCTP, dGTP. The raw material for new DNA.
• Taq DNA polymerase: the enzyme that builds new DNA. Taq is purified from the thermophilic bacterium Thermus aquaticus and survives temperatures up to 95 degrees Celsius without denaturing.
• Buffer and magnesium ions to keep the enzyme working.

The cycle (one of typically 25 to 35):

1. Denaturation (94 to 96 degrees Celsius, about 30 seconds). The mixture is heated, breaking the hydrogen bonds between the two DNA strands. The double helix separates into single strands.
2. Annealing (50 to 65 degrees Celsius, about 30 seconds). The temperature is dropped. The primers bind to their complementary sequences on each single-stranded template by base pairing. The annealing temperature is set just below the melting temperature of the primers.
3. Extension (72 degrees Celsius, about 30 to 60 seconds). Taq polymerase binds each primer-template complex and extends the primer by adding dNTPs in the 5' to 3' direction, complementary to the template. By the end of extension, each template has been copied into a new double-stranded DNA molecule.

Outcome. Each cycle doubles the amount of target DNA: 1 to 2 to 4 to 8 to ... After 30 cycles, the target has been amplified about 10**9 times (a billion-fold).

Visualisation. The amplified product is then loaded into a gel (next section) or sequenced.

Strengths. Extreme sensitivity (single molecules can be amplified), speed (a few hours from sample to product), and specificity (primers ensure only the target region is amplified).

Limitations. Sensitivity is also a weakness: contamination with stray DNA can be amplified just as easily as the target. PCR requires knowledge of the target sequence to design primers.

Gel electrophoresis

Gel electrophoresis separates DNA fragments by size. Used to check PCR products, compare DNA profiles, or sort DNA pieces before sequencing.

Setup:

• An agarose gel is prepared as a slab in a tank of conducting buffer.
• DNA samples are loaded into wells at one end of the gel. A coloured loading dye is added to make samples visible during loading.
• An electric field is applied across the gel: wells at the negative (cathode) end, the other end positive (anode).

The principle:

• DNA is negatively charged (phosphate groups in the sugar-phosphate backbone), so it migrates toward the positive electrode.
• The agarose gel is a porous matrix that acts as a molecular sieve.
• Smaller fragments pass through the pores more easily and travel further.
• Larger fragments are caught up in the matrix and travel less far.

Reading the gel:

• After about 30 to 60 minutes, the current is stopped.
• The gel is stained with a fluorescent dye (such as SYBR Safe or ethidium bromide).
• Under UV light, the DNA fragments appear as bands.
• A DNA ladder (a mix of fragments of known sizes) is run in a parallel lane. The sample fragments' sizes are determined by comparing migration distance with the ladder.

The output is a series of bands; each band is a population of fragments of one size. Larger fragments are near the top of the gel (where the wells were); smaller fragments are near the bottom.

DNA profiling

DNA profiling (also called DNA fingerprinting) uses regions of the genome where individuals differ predictably.

The main targets are short tandem repeats (STRs): short DNA sequences (typically 2 to 6 base pairs) repeated multiple times in tandem. The number of repeats at each STR locus varies highly between individuals.

Steps:

1. Extract DNA from the sample (blood, saliva, hair root, semen).
2. Amplify multiple STR loci by PCR using primers flanking each STR. The amplified fragment's length depends on how many repeats are present at that locus on each chromosome.
3. Run on a gel (or capillary electrophoresis for fine resolution).
4. Read the band pattern: each individual produces a unique pattern of band lengths across the chosen STR loci. Most modern profiles use 13 to 24 STR loci, giving a probability of two unrelated individuals matching of around 1 in a billion or less.

Applications:

• Forensic identification. Comparing DNA from a crime scene with suspects' DNA.
• Paternity testing. A child's DNA profile should contain bands from each biological parent. Mismatches at multiple loci exclude paternity.
• Identification of remains in disasters and historical investigations.
• Wildlife forensics. Identifying species or populations from confiscated samples.

Genetic disease diagnosis: PCR plus gel electrophoresis is also used to detect specific disease-causing mutations: amplify the region containing the gene, then check by restriction digest, allele-specific PCR or sequencing.

Other DNA manipulation tools (background)

• Restriction enzymes cut DNA at specific recognition sequences, producing predictable fragment lengths. Used in cloning and historical DNA fingerprinting (RFLP).
• DNA ligase joins two pieces of DNA at compatible ends.
• Cloning vectors (plasmids) carry foreign DNA into bacteria for propagation.
• Sanger sequencing and next-generation sequencing read the DNA base order.
• CRISPR-Cas9 edits the genome at specific sites (covered in Unit 4).

PCR and gel electrophoresis are the workhorses that underlie all of these.

Worked example

A pregnant woman is screened for cystic fibrosis using a PCR-based test for the delta-F508 allele (the most common CFTR mutation). DNA is extracted from a small amniocentesis sample (just a few cells). Primers flanking the CFTR region around codon 508 are added. After 30 cycles of PCR, the target region is amplified about a billion-fold. The PCR product is run on an agarose gel.

A normal allele gives a band at a certain size; the delta-F508 allele has three fewer base pairs (a 3-bp deletion) and gives a band that is 3 bp shorter. Comparing the patient's pattern to control alleles:

• One band at normal size only: homozygous normal.
• One band 3 bp shorter only: homozygous delta-F508 (affected with CF).
• Two bands at both sizes: heterozygous carrier.

The combination of PCR (sensitive amplification of a small sample) and gel electrophoresis (sensitive separation of similar-sized fragments) makes diagnosis fast and reliable.

Common traps

Saying PCR "creates new genes". PCR copies existing DNA. It does not create new sequences.

Calling Taq "any DNA polymerase". Taq is special because it is heat-stable. Ordinary DNA polymerase would denature at the high temperatures used for denaturation each cycle.

Forgetting primers. Without two specific primers, PCR has no idea what to copy. Primer design is the most important step in setting up a PCR.

Saying "gel electrophoresis separates DNA by charge". All DNA fragments have the same charge-to-mass ratio (one negative charge per nucleotide). Separation in the gel is by size, mediated by the matrix.

Loading at the positive end. DNA loads at the negative end and migrates toward the positive electrode.

Confusing PCR with sequencing. PCR amplifies; sequencing reads the base order. They are often done in sequence (PCR first, then sequencing of the product), but they are different techniques.

In one sentence

PCR amplifies a chosen DNA region a billion-fold by repeated cycles of denaturation (heat the DNA apart), annealing (primers bind their complementary sequences) and extension (heat-stable Taq polymerase copies the strand using free dNTPs), and gel electrophoresis separates the resulting fragments by size as they migrate from the negative loading well toward the positive electrode through an agarose matrix; combined, PCR and gel electrophoresis enable DNA profiling (using short tandem repeats), forensic identification, paternity testing and genetic disease diagnosis.