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?

The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously Signup and view all the answers

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

Guanine pairs with Cytosine through three hydrogen bonds Signup and view all the answers

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

To synthesize DNA by adding nucleotides. Signup and view all the answers

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

The dideoxynucleotide that terminated synthesis. Signup and view all the answers

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

1983 Signup and view all the answers

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

It amplifies a specific DNA sequence to create multiple copies. Signup and view all the answers

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

Invention of the polymerase chain reaction. Signup and view all the answers

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

They proposed the DNA double helix model. Signup and view all the answers

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

Discovery of the first DNA polymerase. Signup and view all the answers

What is the primary purpose of PCR in forensic science?

To amplify target DNA from small samples Signup and view all the answers

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

Variable Number Tandem Repeat Signup and view all the answers

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

Epithelial cells Signup and view all the answers

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

It varies among individuals and aids in identification Signup and view all the answers

In PCR, what role do primers play?

They bind to specific sequences of DNA Signup and view all the answers

When were VNTRs first utilized for DNA profiling?

1984 Signup and view all the answers

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

A variant form of a gene Signup and view all the answers

What is the purpose of separating DNA fragments electrophoretically?

To analyze the size and length of the fragments Signup and view all the answers

In the context of DNA profiling, what are STRs?

Short Tandem Repeats Signup and view all the answers

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

It is derived from Thermus aquaticus, which can survive high temperatures. Signup and view all the answers

What is the role of primers in the PCR process?

To provide a starting point for DNA synthesis. Signup and view all the answers

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

94°C Signup and view all the answers

How long is a typical DNA primer used in PCR?

20-24 nucleotides Signup and view all the answers

What cycles are generally performed in the PCR process?

25-30 cycles Signup and view all the answers

What is the function of dNTPs in the PCR process?

To serve as substrates for DNA synthesis. Signup and view all the answers

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

50°C-65°C Signup and view all the answers

Where was the bacterium Thermus aquaticus isolated from?

A hot spring in Yellowstone National Park. Signup and view all the answers

What is essential for the PCR process to function correctly?

Very precise temperature control. Signup and view all the answers

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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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.