1What is the goal of Sanger sequencing?
Determine the nucleotide sequence of a DNA strand.
2How do ddNTPs differ from dNTPs?
ddNTPs lack a 3′-OH; dNTPs have one.
3Why do ddNTPs stop DNA synthesis?
Without a 3′-OH, another nucleotide cannot be added.
4Why are both dNTPs and ddNTPs needed?
dNTPs extend DNA; ddNTPs terminate strands at specific bases.
5Why does Sanger sequencing use one primer?
To produce fragments extending from one defined starting point in one direction.
6Why does PCR use two primers?
To amplify DNA between two defined sites on opposite strands.
7How are ddNTPs detected in modern Sanger sequencing?
Each ddNTP type has a distinct fluorescent label.
8What is a chromatogram?
A graph of fluorescent peaks representing DNA bases in sequence order.
9What does each chromatogram peak represent?
A detected DNA fragment ending in a labeled nucleotide.
10How is a Sanger chromatogram generated?
Fragments are separated by capillary electrophoresis; fluorescence identifies terminal bases.
11Why do shorter Sanger fragments appear first?
They travel through the capillary faster than longer fragments.
12How is the DNA sequence read from a chromatogram?
Read peaks from left to right, identifying each base by its color.
13What does a clean, single chromatogram peak suggest?
One predominant base was detected at that position.
14What is BLAST used for?
Find similar DNA or protein sequences in a database.
15What does BLAST stand for?
Basic Local Alignment Search Tool.
16How does BLAST identify sequence matches?
It finds local similarities between a query and database sequences.
17What is the query sequence in BLAST?
The DNA or protein sequence submitted for comparison.
18What does a high BLAST alignment score suggest?
A stronger sequence alignment under the scoring system.
19What does a low BLAST E-value indicate?
The match is less likely to occur by chance.
20What does 100% sequence identity mean?
All aligned positions match; it does not prove the organisms are identical.
21What is an antibody?
A protein that specifically binds an antigen.
22What is the basic structure of an antibody?
A Y-shaped protein with two heavy and two light chains.
23What do antibody variable regions do?
Bind specific epitopes on an antigen.
24What do antibody constant regions do?
Provide structural features and interact with other immune components.
25What is an antigen?
A substance specifically recognized by an antibody or immune receptor.
26What is an epitope?
The specific part of an antigen recognized by an antibody.
27Which cells produce antibodies?
Plasma cells, which develop from activated B lymphocytes.
28What does ELISA stand for?
Enzyme-linked immunosorbent assay.
29What is the purpose of an ELISA?
Detect or measure a target antigen or antibody using enzyme-linked detection.
30What is the key feature of direct ELISA?
An enzyme-linked primary antibody binds directly to the target antigen.
31What is the key feature of indirect ELISA?
An unlabeled primary antibody binds the target; an enzyme-linked secondary antibody detects it.
32What is the key feature of sandwich ELISA?
A capture antibody binds the antigen, which is detected by another antibody.
33What is the role of the capture antibody in sandwich ELISA?
Immobilize the target antigen in the well.
34What is the role of an enzyme-linked antibody in ELISA?
Generate a detectable signal through an enzyme-substrate reaction.
35What does a color change in ELISA indicate?
Enzyme activity; under suitable conditions, stronger color indicates more target.
36Why are wash steps important in ELISA?
Remove unbound material to reduce background signal.
37What is the purpose of the indirect ELISA antigen-coating step?
Attach antigen to the well surface.
38What is the purpose of the blocking step in indirect ELISA?
Cover unoccupied well surfaces to