DNA Sequencing

What are the two main DNA sequencing methods compared in the lecture?

Chemical sequencing (Maxam/Gilbert) and chain termination sequencing (Sanger).

What is the primary goal of DNA sequencing?

To determine the sequence of DNA and map approximately 20,000-25,000 genes.

What significant milestone in DNA sequencing was completed in 2003?

The sequencing of 2.91 billion base pairs of the human genome.

What percentage of the human genome encodes for protein production?

Less than 2%.

What are repetitive sequences in the human genome often referred to as?

'Junk DNA'.

What is the significance of single nucleotide polymorphisms (SNPs)?

They represent single base pair DNA differences occurring about once every 100-300 bases.

What are the two manual methods of DNA sequencing mentioned?

Maxam/Gilbert chemical sequencing and Sanger chain termination sequencing.

What is the main advantage of automated fluorescent sequencing?

It reduces background noise and eliminates the need for labeling multiple primers.

What is the role of dideoxynucleotides (ddNTPs) in chain termination sequencing?

They terminate DNA synthesis when incorporated, preventing further elongation of the DNA strand.

How does Maxam-Gilbert sequencing determine the sequence of DNA?

By breaking ssDNA at specific nucleotides and separating fragments on a denaturing polyacrylamide gel.

What is the significance of the order of nucleotide bases in the human genome?

The order is 99.9% identical across all individuals, with differences impacting disease.

What is the purpose of using a labeled primer in sequencing reactions?

To identify the starting point for DNA synthesis and enable detection of the synthesized fragments.

What are the alternative sequencing methods mentioned?

Pyrosequencing and bisulfite sequencing.

What are the components involved in chain termination sequencing?

A primer, template, dideoxynucleotides, and labeled nucleotides.

What is the significance of the ongoing analyses and publications in genomics?

They contribute to the understanding of gene functions, many of which remain unknown.

What is the impact of repetitive sequences on the genome?

They can reshape the genome by rearranging it, creating new genes or modifying existing ones.

What does the term 'human haplotyping' refer to?

The process of mapping genetic variations in human populations.

What are the applications of DNA sequencing in medicine?

Mutation detection, confirmation of polymorphisms, resistance testing, and genotyping.

What is the role of bioinformatics in DNA sequencing?

To analyze and interpret the vast amounts of data generated by sequencing technologies.

What is the significance of the 3' OH group in DNA synthesis?

It is necessary for the addition of nucleotides; ddNTPs lack this group, causing chain termination.

What are the limitations of Maxam-Gilbert sequencing?

It is not practical for high throughput sequencing of long fragments and involves hazardous chemicals.

What is the role of ddNTPs in DNA sequencing?

ddNTPs lack an OH group at the 3' carbon, causing elongation to stop when added.

What happens if the concentration of ddNTPs is too high?

Polymerization will terminate early along the template.

What happens if the concentration of ddNTPs is too low?

No termination will occur, resulting in incomplete sequencing.

What is a sequencing ladder?

A four-lane gel that shows DNA fragments corresponding to each ddNTP (A, C, G, T).

How are sequencing gels read?

From the bottom to the top, indicating the smallest to largest fragments.

What is cycle sequencing?

A chain termination sequencing method performed in a thermal cycler using heat-stable DNA polymerase.

What is the purpose of using fluorescent dyes in sequencing?

Fluorescent dyes allow for the identification of nucleotides by color, enabling all reactions to occur in a single tube.

What is pyrosequencing?

A sequencing method that generates a light signal through the release of pyrophosphate (PPi) during nucleotide addition.

What is the significance of bisulfite sequencing?

It is used to detect methylation in DNA by converting cytosine to uracil, while leaving methylated cytosine unchanged.

What is the main advantage of dye terminator sequencing?

It allows for the labeling of sequencing ladders at the 3' ends of chains, enabling simultaneous reactions.

What is the role of electrophoresis in DNA sequencing?

It separates DNA fragments by length, allowing for the determination of nucleotide sequences.

What is the purpose of a stop buffer in sequencing?

To halt the replication-like process after a specified time.

What are the key components of a sequencing reaction?

Template DNA, primer, dNTPs, ddNTPs, and DNA polymerase.

What does the term 'chain termination' refer to in DNA sequencing?

It refers to the process where the addition of ddNTPs stops the elongation of the DNA strand.

What is the difference between dye primer and dye terminator sequencing?

Dye primer sequencing labels the 5' ends of chains, while dye terminator sequencing labels the 3' ends.

What is the function of the laser in capillary electrophoresis?

To activate fluorescent tags for detecting DNA fragments.

What is the purpose of removing excess dye terminators after sequencing?

To ensure that only the labeled sequencing fragments are analyzed.

How does computational biology relate to DNA sequencing?

It involves the storage, organization, and analysis of vast amounts of sequencing data.

What is the significance of detecting mutations in DNA sequencing?

It helps identify genetic diseases and understand their underlying mechanisms.

What is the expected outcome of comparing treated and untreated sequences in bisulfite sequencing?

To determine which cytosines are methylated based on the differences in sequences.

What is the role of ATP in pyrosequencing?

ATP is generated from PPi and is used to produce light in the presence of luciferase.

What is the importance of using a heat-stable DNA polymerase in cycle sequencing?

It allows for repeated denaturation and synthesis cycles without losing enzyme activity.

What does an electropherogram display?

It shows the results of DNA sequencing, including peak patterns that reflect mutations or sequence changes.

What is the role of the sequencing buffer in DNA sequencing?

It provides the necessary ionic environment for the enzymatic reactions during sequencing.

What is the significance of using a single primer in cycle sequencing?

It ensures that only one strand of DNA is copied, simplifying the sequencing process.