6.3 Manipulating Genomes Flashcards

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1
Q

What did Fred Sanger do in terms of DNA sequencing? Using?

A

-Formed one way to sequence DNA using a single strand
-using nucleotides, DNA polymerase and electrophoresis.

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2
Q

First step in Fred Sanger DNA sequencing? 📍

A
  • Find location of desired gene on DNA strand.
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3
Q

Second step in Fred Sanger DNA sequencing? ⛓️

A
  • break the single stand of DNA
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4
Q

Third step in Fred Sanger DNA sequencing? 👈🏾

A
  • Insert broken DNA to plasmid DNA.
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5
Q

Fourth step in Fred Sanger DNA sequencing? 🦠
Then 😢😷 I?

A
  • Insert recombinant plasmid DNA into bacteria and allow it to replicate.
  • Then Isolate recombinant plasmid DNA to be sent to sequencing.
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6
Q

What is the 4 things that need to be added to DNA sequencing plate? P,B,T,P

A

-DNA Polymerase
-Free DNA bases
-Terminator base
- DNA primer

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7
Q

What temperatures are involved in sequencing in order? 4

A

96, 50, 60, 96

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8
Q

What happens at 96 degrees?
-when does it happen throughout sequencing?

A

The 2 strand DNA separates into 1 strand
-happen at beginning
-happened at end when separating the new stand of DNA from template original recombinant plasmid DNA stand.

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9
Q

What happened at 50 degres?

A
  • Primer binds to plasmid on the recombinant plasmid DNA strand.
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10
Q

What happens at 60 degrees in DNA sequencing?

A

-DNA Polymerase binds the free bases to match DNA template. DNA polymerase is removed.

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11
Q

How are terminator bases modified?

A
  • Modified with coloured fluorescent tags
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12
Q

Why does the process repeat?

A

To get different lengths of DNA fragments.

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13
Q

In electrophoresis, how is DNA pulled through capillary tube gel?
-charge

A

DNA is negatively charge an electric current is added and DNA moves through gel.

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14
Q

And electrophoresis, what is at the end of the capillary tube that allows us to know og DNA sequence?

A

-there is a laser that detect determinator base and record the colour of the base.

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15
Q

What does the colour in electrophoresis DNA sequencing (laser) tell us?

A

-they relate to the base letter. Therefore, we are able to know the sequence of DNA.

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16
Q

What is Pyrosequencing?
- think of mRNA
- complimenting

A

Synthesising a single strand DNA. Complementing the strand to be sequenced

17
Q

What is a nebuliser?
- 🖥️

A

Used to mechanically cut DNA with into fragments

18
Q

What are activated nucleotides?
- what are the examples?

A

-A nucleotide with two extra phosphate groups
- TTP,ATP, CTP, GTP

19
Q

What is Apyrase?
- Unincorporated A N

A

Unincorporated activated nucleotides are degraded and reaction starts again with another nucleotides

20
Q
  1. (Pyrosequencing) what happens to the large piece of DNA?
    -what equipment used?
A
  • large sequence of DNA is cut
  • using nebuliser
21
Q
  1. (Pyrosequencing) What happens after fragments are formed?
A

The DNA is degraded to a single strand (ssDNA)

22
Q

Define what ssDNA is?

A

Single strand DNA

23
Q
  1. (Pyrosequencing) what happens after ssDNA is made?
    -SP
A
  • sequencing primer is added to ssDNA.
24
Q
  1. (Pyrosequencing) when sequencing primer is added what is the DNA incubated with? 6
    - poly.., ATP s (enzyme), luciferase, apyrase, APS, AN
A
  • DNA polymerase
  • ATP Sulfurylase
  • Luciferase
  • Apyrase
  • Adenosine 5’ phoshosulfste (APS)
  • One Activated Nucleotide
25
Q

5.(Pyrosequencing) what happens after incubation what does the activated nucleotides do?

A
  • one activated nucleotide incorporates to complementary strand sequenced by template.
26
Q
  1. (Pyrosequencing) what happens after nucleotide is added?
A
  • the two extra phosphoryl are released as 2 phosphate (ppi)
27
Q
  1. (Pyrosequencing) what happen when phosphate is released?
    -prescience of…
    - the enzyme …
    - converts the ..(before product) to ..( end product)
A
  • In the presence of APS
  • The enzyme ATP Sulfurylase
  • Converts Phosphate to ATP
28
Q
  1. (Pyrosequencing) what happened after we made ATP. -Luciferin
    - in the presence of..
    - the enzyme..
    - converts lu… to Oxy…
A
  • in the presence of ATP
  • the enzyme lusiferase
  • converts lucifern to oxylucifern
29
Q
  1. (Pyrosequencing) after oxyluceferin is made what then happens
    HINT - it creates.. (end result)
A

It creates photons of light

30
Q
  1. (Prysequencing) what happens at the end of the process to get more photons of light
A
  • Repeat the process with other activated nucleotides.
31
Q

What was the human genome?
-scientists predicted 1…
-but 24..

A
  • Scientist predicted there was 100,000 gene but there really was 24000.
32
Q

What are 3 similarities with genome of other individuals in different species?
-chimpanzee
-pigs
>similar to h..
> treat

A
  • Share 99% of genes with chimpanzees.
  • Pigs and humans have similar insulin genes.
  • Pig insulin used to treat patient with diabetes.
33
Q

What did sequencing DNA help confirm in evolutionary relationship?
- Comparing

A
  • Comparing organisms genomes thought to be closely related helped confirm evolutionary relationships.
34
Q

Where do we get DNA from extinct animals?
- B, T

A

-Bones
-Teeth

35
Q

Due to random mutations 3 million places of our DNA is not shared. What is this due to?
- random mutations called su..
- what are the areas called✂️

A

-Random mutations such as substitutions
- These substitutions occur on areas called (SNPs).

36
Q

What are DNA probes used in?
What are they used to do?
What is a probes special quality?Flo🧪

A

-PCR
-They are complementary to known dna sequences used to locate mutant gene
-Fluorescence