Chain Termination DNA Sequencing Flashcards

You may prefer our related Brainscape-certified flashcards:
1
Q

What is Chain Termination DNA Sequencing?

A

A method of DNA sequencing meant for amplification of the DNA fragment to be sequenced by DNA polymerase and incorporation of modified nucleotides – specifically, dideoxynucleotides (ddNTPs)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Step 1

A

Obtain a patient sample (HAS TO BE BACTERIAL)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

In DNA sequencing, we will be looking at the __________

A

16S rRNA gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is the 16S rRNA gene?

A

Ribosomal RNA, a small subunit of prokaryotic ribosomes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

ALL bacteria have _______

A

16s rRNA gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

__________ are unique to each bacterial species

A

Variable regions

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

_____________ do not change between species.

A

Conserved regions

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Make many copies of ______________using ____________ → Necessary to copy __________

A

16S rRNA gene variable regions (billions of copies) , Polymerase Chain Reaction (PCR), target sequence.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

First PCR requires _______, ________, _______, ________, ________, (x2)

A

template DNA, buffer-pH, polymerase-enzyme, dNTPs, primer (x2)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Primer

A

Short strand of DNA that binds to DNA surrounding target sequence. Bind to control regions to amplify variable

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Primers are set down __________.

A

antiparallel

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Polymerase will extend off the ____ end and make a copy, using _________ as template.

A

3’, original

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

The ___ and ___ indicate which part is exposed

A

3’, 5’

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Step 2

A

Run PCR- Make copies of 16s rRNA gene (Variable region, flanked by conserved regions)
a) Primers bind to left and right sequence of interest to amplify variable region
b) Use primers 2x complimentary to conserved region
c) Make billions of copies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Step 3

A

PCR x1
a) add ddNTPs (nucleotides- sugar, phosphate and base- don’t have
3’OH) (Dideoxy- lacking 2 oxygen)
b) DNA polymerase pulls bases onto sequence, some fall off, some create sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Step 4

A

Fragment mixture run through capillary tube, DNA is seperated by size (gel electrophoresis)

17
Q

Step 5

A

DNA sequence interpreted, then BLASTed

18
Q

Purpose of Chain termination

A

-Figuring out which bacteria are present (Diagnostics)
-Finding a particular sequence