Podcast
Questions and Answers
What is the primary purpose of DNA sequencing?
What is the primary purpose of DNA sequencing?
- To insert DNA into plasmid DNA.
- To determine the order of bases in a DNA strand. (correct)
- To replicate bacterial cells.
- To cut DNA into smaller pieces.
Terminator bases are chemically altered to allow further base additions by DNA polymerase.
Terminator bases are chemically altered to allow further base additions by DNA polymerase.
False (B)
During the sequencing reaction, what is the purpose of heating the DNA to 96°C?
During the sequencing reaction, what is the purpose of heating the DNA to 96°C?
To separate the DNA into single strands
In the sequencing reaction, DNA polymerase adds unlabeled DNA bases to the target DNA until a ______ base is added.
In the sequencing reaction, DNA polymerase adds unlabeled DNA bases to the target DNA until a ______ base is added.
Match the color code with the corresponding DNA base:
Match the color code with the corresponding DNA base:
Why are DNA fragments of varying lengths created during the sequencing reaction?
Why are DNA fragments of varying lengths created during the sequencing reaction?
In electrophoresis, shorter DNA fragments move through the gel slower than longer fragments.
In electrophoresis, shorter DNA fragments move through the gel slower than longer fragments.
What is the role of plasmid DNA in the beginning stages of the DNA sequencing process?
What is the role of plasmid DNA in the beginning stages of the DNA sequencing process?
After electrophoresis, a ______ detects the color of the terminator bases as they pass by, which is then recorded by a camera.
After electrophoresis, a ______ detects the color of the terminator bases as they pass by, which is then recorded by a camera.
During the sequencing reaction, after the DNA is initially heated to 96°C, what is the next temperature adjustment and its purpose?
During the sequencing reaction, after the DNA is initially heated to 96°C, what is the next temperature adjustment and its purpose?
Flashcards
DNA Sequencing
DNA Sequencing
Determines the exact order of nucleotide bases (A, C, G, T) within a DNA molecule.
Terminator Bases
Terminator Bases
Modified nucleotides that, when incorporated into a growing DNA strand, halt further elongation and are labeled with fluorescent tags.
Electrophoresis in DNA Sequencing
Electrophoresis in DNA Sequencing
Separate DNA fragments by size, using an electric field to move them through a gel matrix.
Principle of Electrophoresis
Principle of Electrophoresis
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Color code for Adenine (A)
Color code for Adenine (A)
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Color code for Cytosine (C)
Color code for Cytosine (C)
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Color code for Guanine (G)
Color code for Guanine (G)
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Color code for Thymine (T)
Color code for Thymine (T)
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Study Notes
- DNA sequencing determines the order of bases in a DNA strand.
- DNA is cut into smaller pieces for sequencing.
- Smaller pieces are inserted into plasmid DNA.
- Plasmids are put into bacterial cells for replication.
- Bacterial cells multiply, creating many DNA copies.
- DNA is isolated from the bacteria and sent for sequencing.
- Isolated DNA is transferred to a plate for the sequencing reaction.
Sequencing Reaction Components
- Free DNA bases (A, C, G, T)
- DNA polymerase enzyme
- DNA primers
- Modified DNA bases (Terminator bases) with fluorescent tags
Terminator Bases
- Labeled with colored fluorescent tags.
- Chemically altered to prevent further base additions.
Sequencing Reaction Steps
- Heated to 96°C to separate DNA into single strands.
- Temperature lowered to 50°C for DNA primers to bind to plasmid DNA.
- Temperature increased to 60°C for DNA polymerase to bind to the primer.
- DNA polymerase adds unlabeled DNA bases to the target DNA.
- DNA polymerase continues until a Terminator base is added.
- Terminator base addition stops DNA polymerase, causing it to fall away.
- Heated to 96°C again to separate the new DNA strand from the original.
- Heating and cooling cycle repeated to create DNA fragments of varying lengths.
- Fragment length depends on when a Terminator base was added.
Electrophoresis
- Fragments are separated by length using electrophoresis.
- A capillary tube is lowered into each well.
- An electrical charge is applied, moving negatively charged DNA molecules.
- Capillary tubes contain a porous gel.
- Shorter fragments move through the gel faster than longer fragments.
- Fragments are arranged by size from shortest to longest.
- A laser makes the Terminator bases light up as they reach the end of the capillary.
- The color is detected by a camera and recorded
Color Codes for Bases
- A: Green
- C: Blue
- G: Yellow
- T: Red
- The sequencing machine records colors as a series of colored blocks
- Colors are converted into letters to determine the DNA sequence.
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Description
Learn about DNA sequencing: how DNA strands are prepared, fragmented and amplified using plasmids and bacterial cells. The sequencing reaction involves free DNA bases, DNA polymerase, primers, and terminator bases to determine the order of bases in a DNA strand.
