CB Lab 7

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Questions and Answers

What is the starting material for RT-PCR?

Messenger RNA (correct)

Plasmid DNA

Genomic DNA

Complementary DNA

Which of the following is a key application of RT-PCR?

Sequencing of plasmid DNA

Detection of DNA mutations

Amplification of genomic DNA

Analysis of tissue-specific gene expression (correct)

Which reagent is essential for synthesizing cDNA from RNA?

DNA polymerase

RNase A

Oligo (dT)n

Reverse transcriptase (correct)

How does RT-PCR contribute to gene expression analysis?

By comparing RNA levels of genes across tissues Signup and view all the answers

What process differentiates RT-PCR from standard PCR?

RT-PCR initiates with RNA instead of DNA Signup and view all the answers

What is one key difference between genomic DNA and cDNA used in PCR?

Genomic DNA includes introns, while cDNA does not. Signup and view all the answers

Which of the following is an application of RT-PCR?

Analyzing gene expression levels in various tissues. Signup and view all the answers

What is the role of the enzyme Taq Polymerase in the cDNA synthesis process?

It amplifies DNA during the PCR process. Signup and view all the answers

How does PCR facilitate gene expression analysis?

By converting mRNA to cDNA for quantitative assessment. Signup and view all the answers

Which step in the PCR setup is crucial for allowing primers to bind to the target DNA?

Annealing Signup and view all the answers

Which component is necessary for synthesizing cDNA from RNA?

Reverse transcriptase enzyme Signup and view all the answers

What is the primary application of RT-PCR in gene expression analysis?

To measure RNA levels in a sample Signup and view all the answers

Which step in the PCR program is primarily responsible for denaturing the DNA?

Initial denaturation at 95°C Signup and view all the answers

During the annealing step of the PCR process, what is being facilitated?

Binding of primers to the target DNA Signup and view all the answers

What is the purpose of including Taq DNA polymerase in the PCR setup?

To synthesize new DNA strands Signup and view all the answers

What is the final step of the PCR program and its purpose?

Final Extension: to ensure complete synthesis of DNA Signup and view all the answers

cDNA: Reverse transcriptase (RT): Oligo(dT) primer: RNase H:

Reverse transcriptase (RT): Builds the first cDNA strand. Oligo(dT) primer: Binds to the poly-A tail of mRNA. RNase H: Degrades mRNA. Signup and view all the answers

reverse transcription

using RNA to make DNA Signup and view all the answers

central dogma

dna to rna to prot Signup and view all the answers

pcr by who

kary mulis Signup and view all the answers

pcr

Denaturation (45 sec – 1 min) at 95 ℃ Annealing (45 sec) at 55 ℃ Extension (1 – 1.5 min) at 72 ℃ Signup and view all the answers

what is needed for pcr

Target DNA (template) Primers PCR buffer ( Tris HCl, KCl, Mg2+) dNTPs Taq Polymerase Signup and view all the answers

After each PCR cycle, the amount of DNA doubles. The amount of amplified DNA at the end of N cycles is ?

2^n Signup and view all the answers

designing primers, forward and reverse

Forward primer: binds to the DNA bottom strand Reverse primer: binds to the DNA top strand Signup and view all the answers

Study Notes

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 .

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Description

This quiz covers the targeted amplification of specific DNA sequences using PCR and RT-PCR methods. Students will explore the principles behind these techniques, including the design of gene-specific primers and the significance of reverse transcription in molecular biology.