Lecture 10 - DNA Sequencing and PCR Techniques
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Questions and Answers

What aspect of DNA structure is confirmed by Watson and Crick's model?

  • DNA replication is non-conservative
  • DNA can be synthesized in the 3' → 5' direction
  • DNA strands are parallel
  • DNA strands are anti-parallel (correct)

Which statement describes the direction of new DNA synthesis during replication?

  • New DNA is synthesized in the 3' → 5' direction
  • New DNA is synthesized in both directions simultaneously
  • New DNA is synthesized in the 5' → 3' direction (correct)
  • New DNA synthesis is discontinuous only on the leading strand

In Sanger sequencing, what is the role of dideoxynucleotides (ddNTPs)?

  • They act as terminators for DNA chain elongation (correct)
  • They reduce the overall error rate in DNA sequencing
  • They serve as tools for DNA amplification
  • They enhance the replication of DNA

What is a characteristic feature of the leading and lagging strands during DNA replication?

<p>The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously (C)</p> Signup and view all the answers

What is true about the base pairing in DNA as described by Watson and Crick?

<p>Guanine pairs with Cytosine through three hydrogen bonds (D)</p> Signup and view all the answers

What is the primary function of the polymerase in automated dideoxy sequencing?

<p>To synthesize DNA by adding nucleotides. (C)</p> Signup and view all the answers

What does the dye in each DNA fragment synthesized during automated dideoxy sequencing indicate?

<p>The dideoxynucleotide that terminated synthesis. (C)</p> Signup and view all the answers

In what year was the polymerase chain reaction (PCR) invented?

<p>1983 (B)</p> Signup and view all the answers

Which of the following best describes the role of PCR in molecular biology?

<p>It amplifies a specific DNA sequence to create multiple copies. (D)</p> Signup and view all the answers

Which major advancement in molecular biology is associated with Kary Mullis?

<p>Invention of the polymerase chain reaction. (C)</p> Signup and view all the answers

What is the relationship between Watson and Crick and the study of DNA?

<p>They proposed the DNA double helix model. (B)</p> Signup and view all the answers

What is the significance of Kornberg's work in 1957?

<p>Discovery of the first DNA polymerase. (C)</p> Signup and view all the answers

What is the primary purpose of PCR in forensic science?

<p>To amplify target DNA from small samples (B)</p> Signup and view all the answers

What does VNTR stand for in the context of DNA profiling?

<p>Variable Number Tandem Repeat (A)</p> Signup and view all the answers

Which type of sample is commonly analyzed in forensic science using PCR?

<p>Epithelial cells (C)</p> Signup and view all the answers

What is the significance of the number of repeats in an STR?

<p>It varies among individuals and aids in identification (D)</p> Signup and view all the answers

In PCR, what role do primers play?

<p>They bind to specific sequences of DNA (A)</p> Signup and view all the answers

When were VNTRs first utilized for DNA profiling?

<p>1984 (D)</p> Signup and view all the answers

What does the term 'allele' refer to in genetics?

<p>A variant form of a gene (C)</p> Signup and view all the answers

What is the purpose of separating DNA fragments electrophoretically?

<p>To analyze the size and length of the fragments (A)</p> Signup and view all the answers

In the context of DNA profiling, what are STRs?

<p>Short Tandem Repeats (D)</p> Signup and view all the answers

What is the main reason Taq polymerase is utilized in PCR?

<p>It is derived from Thermus aquaticus, which can survive high temperatures. (B)</p> Signup and view all the answers

What is the role of primers in the PCR process?

<p>To provide a starting point for DNA synthesis. (A)</p> Signup and view all the answers

At what temperature does the denaturation of DNA occur during PCR?

<p>94°C (B)</p> Signup and view all the answers

How long is a typical DNA primer used in PCR?

<p>20-24 nucleotides (D)</p> Signup and view all the answers

What cycles are generally performed in the PCR process?

<p>25-30 cycles (A)</p> Signup and view all the answers

What is the function of dNTPs in the PCR process?

<p>To serve as substrates for DNA synthesis. (A)</p> Signup and view all the answers

Which temperature stage allows for the hybridization of primers in the PCR process?

<p>50°C-65°C (B)</p> Signup and view all the answers

Where was the bacterium Thermus aquaticus isolated from?

<p>A hot spring in Yellowstone National Park. (A)</p> Signup and view all the answers

What is essential for the PCR process to function correctly?

<p>Very precise temperature control. (B)</p> Signup and view all the answers

Flashcards

DNA sequencing

The technique used to determine the order of nucleotides in a DNA sequence.

Dideoxynucleotide (ddNTP)

A modified nucleotide that lacks a 3'-hydroxyl group, which prevents further DNA elongation during replication.

Polymerase Chain Reaction (PCR)

The process of copying a specific DNA sequence many times, amplifying its amount.

Gel Electrophoresis

A method used to detect DNA fragments of different sizes based on their migration speed through a gel under an electric field.

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DNA Replication

The process of copying a DNA sequence using enzymes, building a new complementary strand.

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Automated Dideoxy Sequencing

A technique for determining the sequence of nucleotides in a DNA molecule using fluorescently labeled dideoxynucleotides that terminate DNA synthesis.

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Kary Mullis

The scientist who invented PCR, a transformative tool in molecular biology, awarded the Nobel Prize in Chemistry in 1993.

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Watson and Crick DNA Model

The foundational model of DNA structure, discovered in 1953 by Watson and Crick, explaining the double helix configuration.

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Kornberg DNA Polymerase

The first DNA polymerase enzyme discovered in 1957, a crucial element for DNA replication and PCR.

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Taq Polymerase

A heat-stable DNA polymerase enzyme isolated from a bacterium that thrives in hot springs. PCR relies on this enzyme's ability to withstand high temperatures without denaturing.

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Target DNA

A double-stranded DNA molecule containing the specific region of interest that needs to be amplified.

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Primers (in PCR)

Short, single-stranded DNA sequences designed to bind specifically to the ends of the target DNA sequence, providing starting points for DNA replication.

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Denaturation (in PCR)

The process of separating the double-stranded DNA into two single strands by applying heat, which breaks the hydrogen bonds between the base pairs.

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Annealing (in PCR)

The process where primers bind to the complementary sequences on the single-stranded DNA target molecules, initiating DNA replication.

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Extension (in PCR)

The process where Taq polymerase extends the primers along the single-stranded DNA templates, building new complementary strands.

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PCR Cycle

A repeated series of three steps: Denaturation, Annealing, and Extension. Each cycle doubles the amount of target DNA, resulting in exponential amplification.

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PCR Machine

A laboratory instrument that precisely controls the temperature at different stages of the PCR process, making it possible for the cycle to repeat many times.

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What is Polymerase Chain Reaction (PCR)?

A process that uses enzymes to amplify specific DNA regions, creating millions of copies from a small sample. It has applications in forensics, medicine, and research.

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What are Variable Number Tandem Repeats (VNTRs)?

Small, repeated sequences of DNA bases (typically 3-5 base pairs) which vary in number between individuals. These variations can be used to create unique DNA profiles for identification.

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What are Primers in PCR?

Short, single-stranded DNA sequences that are complementary to the target DNA. They act as starting points for the DNA polymerase enzyme during PCR.

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What is the role of DNA profiling in forensic science?

The use of DNA profiling based on VNTRs (now called STRs), which are inherited from parents, to identify individuals in legal investigations.

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How does PCR help in blood-type analysis?

A forensic technique that uses PCR to amplify very small samples of DNA, allowing for identification from tiny traces of blood or other biological material.

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Who discovered VNTRs and how did they revolutionize forensic science?

Sir Alec Jeffreys, a British geneticist, discovered VNTRs in 1984 and pioneered their application in DNA fingerprinting. He paved the way for modern forensic science and personalized medicine.

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How do you separate and visualize DNA fragments?

The process of separating DNA fragments based on their size using an electric field. Smaller fragments travel faster through a gel matrix, allowing for the visualization of different DNA sizes.

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How is PCR used in forensic science?

Forensic science uses PCR and DNA profiling to analyze evidence and identify suspects in criminal investigations.

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How are VNTRs used to identify individuals?

Chromosomes come in pairs, one from each parent. During PCR, primers amplify specific regions of DNA on homologous chromosomes, revealing variations in the number of repeats between the two copies.

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Study Notes

DNA Sequencing and PCR Techniques

  •  Lecture 10, 2024, Part 1.
  •  Focuses on DNA sequencing techniques and PCR.
  •  Image shows a DNA sequence chromatogram.

DNA Replication

  •  DNA replication is semi-conservative.
  •  DNA strands are anti-parallel.
  •  Watson-Crick base pairing is crucial.
  •  DNA synthesis is semi-continuous, with a leading and lagging strand.

DNA Sequencing Methods

  •  Walter Gilbert (Harvard): Partial chemical degradation of radiolabeled DNA.
  •  Frederick Sanger (MRC Labs, Cambridge, 1970s): Enzyme-mediated incorporation of dideoxynucleotides into newly-replicated DNA.

Sanger Sequencing - Dideoxynucleotides

  •  Relies on the incorporation of dideoxynucleotides into newly-replicated DNA.
  •  Dideoxynucleotides lack a 3' hydroxyl group.
  •  This stops DNA chain elongation.

Dideoxynucleotide as Terminator

  •  Dideoxynucleotides (ddNTPs) have no 3'-OH group.
  •  Incorporation of ddNTPs halts DNA synthesis.
  •  This results in a series of DNA fragments of different lengths.

Sanger's Dideoxy Sequencing Method - Principle

  •  Single-stranded template DNA.
  •  Primer complementary to a part of the template.
  •  DNA polymerase.
  •  Normal deoxynucleotides (dNTPs).
  •  Radioactively-labelled ddNTPs.
  •  The result is a nested set of DNA molecules ending in ddA.

Sanger's Dideoxy Sequencing Method - Practice

  •  Template + primer + DNA polymerase + dNTPs.
  •  Add appropriate ddNTPs.
  •  Separate nested fragments based on size by electrophoresis.
  •  Autoradiograph and read sequence from the bottom upwards.

Automated Dideoxy Sequencing

  •  Fluorescent ddNTPs are used instead of radioactively-labeled ddNTPs.
  •  The dye's colour identifies the terminating nucleotide.
  •  Electrophoretic separation and laser detection of fluorescence.

PCR History

  •  1953: Watson and Crick: DNA molecule structure.
  •  1957: Kornberg: First DNA polymerase.
  •  1960s: Khorana: Genetic code deciphered.
  •  1969: Brock: Isolation of Thermus aquaticus.
  •  1971: Khorana's group: Proposed template-primer-polymerase system.
  •  1976: Taq polymerase isolated.

Kary Mullis and PCR

  •  PCR: Powerful molecular biology tool by Kary Mullis (1983).
  •  Nobel Prize in Chemistry.
  •  Amplifies specific DNA sequences.
  •  "Copying machine" for DNA.

PCR Components

  •  Template dsDNA: Target area & sequence knowledge.
  •  Primers: Specific oligodeoxynucleotides.
  •  dNTPs: Normal deoxynucleotides.
  •  Buffer and MgCl2.
  •  Taq polymerase.

Why Taq Polymerase is Useful

  •  Thermus aquaticus, a heat-resistant bacterium.
  •  Taq polymerase can withstand high temperatures.
  •  Essential for PCR cycling.

How PCR Works

  •  Denaturation (94°C): Separates DNA strands.
  •  Annealing (45-65°C): Primers bind to target DNA.
  •  Extension (72°C): Taq polymerase extends primers.

Applications of PCR

  •  Amplifying tiny DNA amounts.
  •  Medical-genetic analysis.
  •  Forensic science (e.g., DNA profiling).
  •  Analyzing archaeological and ancient DNA.

Specific Mutation Creation using PCR

  •  Mutated primers introduce mismatches.
  •  These mismatches are incorporated into the new DNA.

Sequencing of Archaic Hominin Genomes

  •  Nuclear DNA from fossil Neanderthals amplified & compared to modern DNA.
  •  Europeans and Asians share 4-5% of their genes with Neanderthals.
  •  Gene flow is detected from Neanderthals into modern humans, not the reverse.
  •  Suggests male Neanderthal/female human couplings.

Visualizing PCR Reactions

  •  Agarose gel electrophoresis.
  •  DNA fragments separated by size under electricity.
  •  Fluorescent dye marks the DNA.
  •  UV light visualizes DNA fragments.

Multiplex PCR

  •  Identifies specific primer sets at once from different chromosomes.
  •  Combined DNA Index System (CODIS) in forensic science.

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Related Documents

Lecture 10: PCR and Sequencing PDF

Description

This quiz covers key concepts from Lecture 10 on DNA sequencing techniques and PCR. It discusses various methods of DNA sequencing, including the Sanger method, and the important role of dideoxynucleotides in terminating DNA synthesis. Test your understanding of these fundamental molecular biology techniques.

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