CB Lab 7

Lab 7: Targeted Amplification from Genomic DNA and cDNA by PCR

• Lab focused on amplifying specific DNA sequences or genes using PCR and RT-PCR methods.
• First-strand cDNA synthesis was performed by reverse transcription.
• Course: BIOL 3120 - Cell Biology Lab
• Instructor: Rubaia Tasmin
• Date: 10/21/2024

Today's Lab Objectives

• Understand the principles of PCR and RT-PCR.
• Learn to design gene-specific primers (using Arabidopsis ACTIN1 as an example).
• Set up and run PCR reactions.

The Central Dogma of Molecular Biology

• DNA replication (DNA → DNA) is catalyzed by DNA Polymerase.
• Transcription (DNA → RNA) is catalyzed by RNA Polymerase from DNA to mRNA.
• Reverse transcription is from RNA to cDNA via reverse transcriptase.
• Translation (RNA → Protein) converts mRNA into proteins. This process occurs in ribosomes.
• Unusual flows of information are highlighted in diagrams.

Genomic DNA Organization for Protein-Coding Genes

• Genes consist of exons (coding sequences) and introns (non-coding sequences).
• Introns are removed from pre-mRNA during RNA splicing.
• Exons remain in mature mRNA and are translated into proteins.
• Diagrams of gene structure and mRNA processing are included.

Polymerase Chain Reaction (PCR)

• Powerful method for amplifying specific DNA sequences.
• Developed by Kary Mulis (1983).
• Nobel Prize awarded in 1993.
• Includes steps: denaturation, annealing, and extension.
• PCR cycles are repeating steps to amplify specific DNA section.

PCR Steps

• Denaturation: (45 sec - 1 min) at 95°C, DNA strands separate.
• Annealing: (45 sec) at 55°C, primers bind to complementary DNA strands.
• Extension: (1 - 1.5 min) at 72°C, DNA polymerase synthesizes new strands.

Contents of a PCR Reaction

• Template DNA.
• Primers (forward and reverse).
• PCR buffer (Tris HCI, KCl, Mg²⁺).
• Deoxynucleotide triphosphates (dNTPs).
• Taq polymerase.

Designing Primers

• Forward primers bind to the bottom strand of DNA.
• Reverse primers bind to the top strand of DNA.
• Primer sequences are provided from 5' to 3' direction.
• Primers are short pieces of single stranded DNA.
• They bind to specific DNA sequences.

Designing Primers for a DNA Segment in ACTIN1 Gene

• Provide sequences of forward and reverse primers (specified as At-ACTIN-483F & At-ACTIN-D40-R2).

PCR Troubleshooting

• Why do primers anneal to the target DNA? High concentration of primers prevents re-hybridization.
• Why does PCR commonly run for 30 cycles? Primers and dNTPs are consumed.
• Why is product length defined by 5' end? Polymerase may extend beyond primer's boundary.

Reverse Transcription (RT) and cDNA synthesis

• cDNA is the complementary DNA copy of mRNA.
• RT-PCR uses mRNA converted to cDNA as a starting material.
• RT reaction involves reverse transcriptase and oligo(dT)n to make cDNA sequence from mRNA.
• RNaseH removes mRNA.

Difference between PCR and RT-PCR

• PCR uses gDNA, RT-PCR uses cDNA which is created from RNA.
• gDNA has introns, cDNA does not.
• RT-PCR is needed to study gene expression.

PCR Reaction Setting-up (A)

• Components: 10X Taq buffer, PCR nucleotides mix, forward and reverse primers, Taq DNA poymerase, genomic DNA and volumes.

PCR Reaction Setting-up (B)

• Components: 10X Taq buffer, PCR nucleotides mix, forward and reverse primers, Taq DNA poymerase, cDNA and volumes.

PCR Program

• Initial Denaturation: 95°C for 2 minutes.
• Denaturation: 94°C for 1 minute.
• Annealing: 59°C for 1 minute.
• Extension: 72°C for 50 seconds.
• Repeat steps 2-4 for 29 cycles.
• Final Extension: 72°C for 10 minutes.
• Storage: 4°C .