Topic 8 - Applications of PCR: Detailed Study Guide

Topic 8: Learning Aims and Overview

Core Objectives: * Explain the fundamental components of a Polymerase Chain Reaction ( $PCR$ ) reaction. * Explain the three-step process of $PCR$ : denaturation, annealing, and extension ( $elongation$ ). * Discuss medical applications of $PCR$ , specifically regarding diagnostics and screening. * Discuss infectious disease applications of $PCR$ . * Discuss forensic applications of $PCR$ , including genetic fingerprinting, Restriction Fragment Length Polymorphism ( $RFLP$ ), Variable Number Tandem Repeat ( $VNTR$ ), and Short Tandem Repeat ( $STR$ ). * Discuss research applications of $PCR$ .

The PCR Process and Components

The Three Steps of PCR: * 1. Denaturation: The double-stranded $DNA$ template is heated to separate into two single strands. * 2. Annealing: Temperature is lowered to allow primers to bind to their complementary sequences on the single-stranded $DNA$ . * 3. Elongation (Extension): $Taq$ polymerase adds nucleotides to the primers, synthesizing a new $DNA$ strand. * Cycle Progression: The sequence of steps ( $1, 2, \text{ and } 3$ ) is repeated multiple times, leading to the exponential growth of the short target product.
Essential PCR Components: * Template DNA: The original $DNA$ molecule containing the target sequence to be amplified. * Primers: Two short $DNA$ sequences complementary to the $3'$ ( $three prime$ ) ends of each of the sense and anti-sense strands of the target $DNA$ . Primers can be designed for any specific gene. * dNTPs (Deoxynucleotide Triphosphates): The building blocks (A, T, C, G) used by the polymerase to synthesize new $DNA$ . * Taq Polymerase: A heat-stable $DNA$ polymerase used to assemble the new $DNA$ strands. * Equipment: The reaction typically takes place in a Thermocycler (e.g., brands like Biometra).

Interpreting PCR and Gel Electrophoresis Results

Agarose Gel Electrophoresis: * Used to visualize the results of $PCR$ amplification. * Ladder/Molecular Weight Marker: Used to determine the size of the synthesized $DNA$ bands. For example, a $122\,bp$ band or a $1\,kb$ ladder may be used for reference. * Visualization: Gels are stained with Ethidium Bromide and photographed under $UV$ light. * Controls: * Positive Control: Used to ensure the $PCR$ conditions were adequate for amplification. * Negative Control: Used to ensure no contamination is present; no amplification should be observed. * Example Analysis: In a test of three separate tissues using two primers, if Tissue #1 lacks a band while Tissue #2 and #3 possess it, it indicates the specific gene is not expressed or present in Tissue #1.

Medical Applications: Diagnostics and Screening

Genetic Disease Diagnostics: * Examples: Hemophilia and Cystic Fibrosis. * Mechanism: Primers are used to amplify only the mutated portion of a gene. If a product is created, the mutated gene is confirmed to be present. * Cancer Related Genes: The $HaloPlex^{TM}$ PCR $BRCA$ Panel Kit is used for efficient analysis of regions of interest in breast cancer susceptibility genes $BRCA1$ and $BRCA2$ .
Genetic Screening Types: * Prenatal Testing: Screening for genetic diseases offered to women during pregnancy. * Newborn Screening: Carried out routinely in hospitals worldwide for conditions like Cystic Fibrosis. * Carrier Screening: Used to determine if individuals carry a mutation that does not affect their health but could affect their future children (e.g., autosomal recessive inheritance).
Preimplantation Genetic Diagnosis (PGD): * A technique to test embryos for specific genetic disorders before transfer during $IVF$ . * Testing is typically performed at the Day $3$ , $6-8$ cell stage of the embryo. * This allows the selection of only unaffected embryos for transfer.

Case Study: Cystic Fibrosis (CF)

Overview: $CF$ is characterized by thick, sticky mucus that blocks airways and pancreatic/bile ducts.
Genetic Basis: Caused by mutations in the $CFTR$ gene located on Chromosome $7$ .
Cellular Mechanism: * Normal CFTR Channel: Moves chloride ( $Cl^-$ ) ions to the outside of the cell. * Abnormal CFTR Channel: Fails to move chloride ions, causing sticky mucus to build up outside the cell.
Newborn Screening Procedure: 1. Blood specimen collected on a card. 2. Punch a hole from the dried blood spot. 3. Proteinase K Digestion and lysis (using tools like $ZR$ BashingBead Lysis Tube). 4. Purification steps: Bind, Wash, Elute. 5. Result: Ultra-pure $DNA$ ready for $PCR$ .
Inheritance: Autosomal recessive. If both parents are carriers: * $25\%$ chance the child is Normal. * $50\%$ chance the child is a Carrier. * $25\%$ chance the child has Cystic Fibrosis.

Infectious Disease Applications

Detected Pathogens: Tuberculosis ( $TB$ ), $HIV$ , Gonorrhea, and Chlamydia. * Chlamydia Detection: Chlamydia is a bacterial $STD$ . $PCR$ is preferred over Gram staining or culturing because of its sensitivity. * Amplicon Size: For Chlamydia, a representative $2\%$ agarose gel might show a target amplicon band at $260\,bp$ .

Forensic Applications and Genetic Variation

Polymorphism: The presence of genetic variation within a population, involving two or more variants of a $DNA$ sequence. * Single Base Pair Variation: The most common type of polymorphism. * Nucleotide Diversity: Estimated to be $0.1\%$ to $0.4\%$ of base pairs in humans. * Quantification: Approximately $3$ million nucleotide differences in a human genome of approximately $3$ billion nucleotides.
Evidence Techniques: * RFLP (Restriction Fragment Length Polymorphism): Exploits variations in homologous $DNA$ sequences at restriction enzyme sites. It requires a significantly larger blood sample compared to $PCR$ . * VNTR (Variable Number Tandem Repeat): Short nucleotide sequences organized as tandem repeats. Variants act as inherited alleles used for parental or personal identification. * STR (Short Tandem Repeat): Involves tracking the number of repeats at specific loci (e.g., Man 1 has $5$ repeats, Man 2 has $6$ , Man 3 has $7$ ). * CODIS: Uses $13$ Core $STR$ Loci with specific chromosomal positions (e.g., $TPOX$ , $CSF1PO$ , $TH01$ , $vWA$ , $D3S1358$ ) for forensic matching.
Legal Case Studies: * Colin Pitchfork (1988): The first conviction resulting from a mass $DNA$ screen. * OJ Simpson Trial: Blood samples matched $OJ$ 's genetic identity, but the defense alleged samples were tainted, mishandled, or switched ("The DNA Wars"), leading to a "not guilty" verdict. * Exonerations: Illinois Governor George Ryan used $DNA$ testing in $1998$ to exonerate $13$ of $25$ death row inmates. Roy Criner was exonerated by $DNA$ testing for a rape and murder charge after initial judicial resistance.

Biological Complexities in DNA Evidence

Microchimerism: Women may carry at least three unique cell populations: their own, their mother's, and their child's.
Chimerism: An individual can effectively be their own twin. Lydia Fairchild was accused of fraud when maternity tests suggested she was not the mother of her own children; she was later found to be a chimera.

Research and Future Directions

Research Tools: Tools like Reverse Transcriptase ( $RT$ ) are used to convert $RNA$ into $cDNA$ for $PCR$ amplification.
PCR vs. Sequencing: * PCR: Looks at alleles, large masses of $DNA$ , and $STR$ sizes resulting in bands. * Sequencing: Identifies each individual letter of the $DNA$ code. * Trend: Whole Genome Sequencing ( $WGS$ ) costs are rapidly reducing, potentially shifting reliance away from traditional $PCR$ for some applications.

Paternity and Parental Testing

Procedure: 1. Treat $DNA$ from Mother, Father, and Child with restriction endonucleases. 2. Separate fragments via gel electrophoresis. 3. Blot to nitrocellulose and probe with labeled $DNA$ . 4. Prepare an autoradiogram to compare bands.
Logic: A child must receive one copy of every genetic marker/allele from each biological parent. Bands in the child's profile must be present in either the mother's or the father's profile.