Omics-C14 Flashcards
DNA has a double-helix structure, proposed by James Watson and Francis Crick in 1953, based on Rosalind Franklin’s X-ray diffraction data. The two strands of the helix run antiparallel to each other, meaning: ____
one strand runs in the 5’ to 3’ direction and the other in the opposite 3’ to 5’ direction.
What is DNA sequencing?
DNA sequencing is the process of determining the exact order of the nucleotides within a DNA molecule.
How is Eukaryotic DNA formed vs. Prokaryotic DNA?
Eukaryotic DNA is linear and tightly packed into chromosomes in the nucleus. The DNA wraps around histone proteins to form nucleosomes. These nucleosomes are further condensed into chromatin and, during cell division, into chromosomes.
Prokaryotic DNA is usually circular and resides in the cytoplasm in a region called the nucleoid. Prokaryotes typically have a single circular chromosome.
Prokaryotic DNA is circular and located in the nucleoid, whereas eukaryotic DNA is linear, tightly packed into chromosomes, and resides in the nucleus.
Explain sanger method of DNA sequencing
The Sanger method of DNA sequencing is a process used to determine the exact sequence of nucleotides (A, T, C, G) in a piece of DNA. Here’s a simple breakdown of how it works:
Steps of the Sanger Method:
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Prepare the DNA:
- The DNA you want to sequence is copied, and this copied DNA is placed into four separate test tubes.
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Add Ingredients:
- In each test tube, you add:
- Normal nucleotides (A, T, C, and G) that help build the DNA strands.
- A special enzyme called DNA polymerase that helps create the DNA copy.
- Modified nucleotides (called ddNTPs) are added. These modified nucleotides are a bit different from normal ones: when they are added to the DNA strand, they stop the DNA from growing any further. These modified nucleotides are labeled with different colors for A, T, C, or G, so you can easily see which one was added.
- In each test tube, you add:
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Building the DNA Strands:
- As the enzyme copies the DNA, it randomly inserts either a normal nucleotide or a modified nucleotide (ddNTP).
- Every time a modified nucleotide is added, the copying stops, so you end up with DNA pieces of different lengths.
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Separating the DNA Fragments:
- The different-sized DNA fragments are then separated by size using a process called gel electrophoresis. Smaller fragments move faster, while larger fragments move slower.
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Reading the Sequence:
- A special machine reads the colors of the modified nucleotides at the end of each fragment to determine which base (A, T, C, or G) is present. By lining up the DNA fragments by size, you can determine the exact sequence of nucleotides in the original DNA.
Simplified Analogy:
Imagine copying a sentence, but you use a special marker that sometimes makes you stop at random letters. You end up with different lengths of the sentence. By organizing the sentences by length and looking at the last letter in each sentence, you can figure out the entire sentence letter by letter.
Key Points:
- The Sanger method is like building a copy of a DNA strand but with occasional stops.
- These stops help identify which nucleotide (A, T, C, or G) was added last in each fragment.
- The method gives you the sequence of nucleotides by reading the color-labeled stops in order.
In summary, the Sanger method works by creating DNA fragments that stop growing at specific nucleotides, allowing scientists to determine the exact sequence of the original DNA strand.
Three Models of Replication:
Conservative Model: The parental DNA remains intact, and an entirely new DNA molecule is formed.
Semi-Conservative Model: Each DNA molecule consists of one parental strand and one new strand (this model is correct).
Dispersive Model:DNA is copied in short sections, and both strands have a mixture of old and new DNA.
Which type of these replications happens in our body
Semi-Conservative Replication:
DNA replication follows the semi-conservative model, where each of the two parental DNA strands serves as a template for new strands.
After replication, each new DNA molecule has one old parental strand and one newly synthesized strand.
What is the process of DNA replication?
Process of DNA Replication:
- DNA replication begins at specific sites called origins of replication, where the two strands of DNA are unwound by enzymes.
- The unwinding creates a replication fork, and each of the two strands serves as a template for the synthesis of a new strand.
- Enzymes like DNA polymerase add nucleotides to the growing DNA strand, matching the bases with their complementary pairs: A pairs with T, and G pairs with C.
The ____ strand is synthesized continuously in the same direction as the replication fork.
The ____ strand is synthesized discontinuously in short segments called Okazaki fragments.
These fragments are later joined together by an enzyme called ____.
leading
lagging
DNA ligase
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Enzymes Involved in Replication:
Helicase: ____.
DNA polymerase: ____.
Primase: ____.
Ligase: ____.
Topoisomerase: ____.
- Unwinds the DNA double helix
- Adds nucleotides to the growing DNA strand
- Synthesizes a short RNA primer to initiate DNA synthesis
- Joins the Okazaki fragments on the lagging strand
- Prevents over-winding of the DNA ahead of the replication fork by making temporary cuts
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RNA primer (Primase):
An RNA primer is a short segment of RNA (ribonucleic acid) that is used to initiate the synthesis of a new DNA strand during DNA replication. It is essential because DNA polymerase, the enzyme responsible for adding nucleotides to the growing DNA strand, cannot start the process on its own. DNA polymerase can only add nucleotides to an existing strand with a free 3’ hydroxyl (OH) group.
Key Points about RNA Primer:
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Synthesis by Primase:
- The enzyme primase synthesizes the RNA primer by laying down a short sequence of RNA nucleotides (usually 5-10 nucleotides long).
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Function:
- The RNA primer provides a starting point with a free 3’ OH group, which is necessary for DNA polymerase to begin adding DNA nucleotides to elongate the new strand.
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Where It’s Used:
- On the leading strand, only one RNA primer is needed at the start of replication.
- On the lagging strand, multiple RNA primers are needed because the strand is synthesized in short sections called Okazaki fragments. Each fragment requires a new RNA primer.
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Removal and Replacement:
- Once DNA synthesis begins, the RNA primer is later removed and replaced with DNA nucleotides by another enzyme, DNA polymerase I. The fragments are then joined together by DNA ligase.
Summary:
The RNA primer acts as a starter sequence for DNA synthesis by providing the necessary 3’ OH group for DNA polymerase to add DNA nucleotides. Without the RNA primer, DNA polymerase would not be able to start the replication process.
DNA polymerase can only synthesize new DNA in the ____ (5’ to 3’ / 3’ to 5’ ) direction.
5’ to 3’
Prokaryotic DNA is circular, and replication occurs ____ (unidirectionally/ bidirectionally) from a single origin.
Okazaki fragments are synthesized on the lagging strand and later joined by DNA ____.
Prokaryotic replication is much ____ (slower/faster) than eukaryotic replication, adding up to 1000 nucleotides per second.
bidirectionally
ligase
faster
DNA replication in Prokaryotes
Steps of Replication:
Initiation:
The process begins at the origin of replication, a specific nucleotide sequence that signals the start of replication.
Helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous bases, forming two replication forks.
Single-strand binding proteins stabilize the separated strands to prevent them from recoiling into a double helix.
Topoisomerase prevents the overwinding of DNA ahead of the replication fork by creating temporary nicks in the DNA helix and then resealing it.
Elongation:
DNA polymerase III is the main enzyme responsible for adding nucleotides to the growing DNA strand. It adds nucleotides in the 5’ to 3’ direction, requiring a free 3’-OH group.
Primase synthesizes short RNA primers to provide the free 3’-OH group for DNA polymerase III to start adding nucleotides.
The leading strand is synthesized continuously in the direction of the replication fork, while the lagging strand is synthesized discontinuously in short fragments called Okazaki fragments.
As the replication fork progresses, more RNA primers are laid down on the lagging strand, and DNA polymerase adds nucleotides to form the Okazaki fragments.
Termination and Cleanup:
After the Okazaki fragments are synthesized, DNA polymerase I removes the RNA primers and fills the gaps with DNA nucleotides.
DNA ligase seals the nicks between the newly synthesized DNA fragments, forming a continuous strand.
Conclusion:
Once replication is completed, the two circular DNA molecules are fully separated, and each daughter cell receives a complete copy of the genome during cell division.
Enzymes Involved:
DNA polymerase III: Synthesizes new DNA strands.
Helicase: Unwinds the DNA helix.
Primase: Synthesizes RNA primers.
DNA polymerase I: Replaces RNA primers with DNA.
DNA ligase: Seals gaps between DNA fragments.
Topoisomerase: Relieves tension from overwinding.
DNA replication in eukaryotes starts at ____ (multiple/single) origins of replication
multiple
there can be up to 100,000 origins in humans
Steps of DNA replication in Eukaryotes
- DNA unwinds at the origin of replication.
- Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
- Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
- Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
- Primase synthesizes RNA primers complementary to the DNA strand.
- DNA polymerase III starts adding nucleotides to the 3’-OH end of the primer.
- Elongation of both the lagging and the leading strand continues.
- RNA primers are removed by exonuclease activity.
- Gaps are filled by DNA pol I by adding dNTPs.
- The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Check the steps see if they are different from prokaryotes
Key Enzymes in Eukaryotic DNA Replication:
DNA polymerase α, δ, and ε: These are the primary enzymes responsible for DNA synthesis. DNA pol α initiates replication by adding a short DNA fragment to the RNA primer, after which pol δ and pol ε take over for elongation.
Sliding clamp protein (PCNA): This holds the DNA polymerase in place during replication.
Telomerase: Involved in replicating the ends of linear chromosomes (telomeres). Telomerase adds repetitive sequences to telomeres to protect important coding DNA from being lost during replication.
Replication Fork and Leading/Lagging Strands:
Similar to prokaryotic replication, DNA is synthesized continuously on the leading strand and discontinuously on the lagging strand (forming Okazaki fragments).
The lagging strand requires multiple primers due to its direction of replication being opposite to the unwinding of the helix.
Telomeres and Telomerase:
Telomeres are repetitive sequences at the ends of chromosomes that protect them from degradation.
Telomerase extends these ends by adding telomere sequences, preventing the loss of essential genetic material during replication. Telomerase is usually active in germ cells and stem cells but not in somatic cells.
