6.3 Manipulating Genomes

Question 1

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

Answer 1

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

Question 2

First step in Fred Sanger DNA sequencing? 📍

Answer 2

• Find location of desired gene on DNA strand.

Question 3

Second step in Fred Sanger DNA sequencing? ⛓️

Answer 3

• break the single stand of DNA

Question 4

Third step in Fred Sanger DNA sequencing? 👈🏾

Answer 4

• Insert broken DNA to plasmid DNA.

Question 5

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

Answer 5

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

Question 6

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

Answer 6

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

Question 7

What temperatures are involved in sequencing in order? 4

Answer 7

96, 50, 60, 96

Question 8

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

Answer 8

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.

Question 9

What happened at 50 degres?

Answer 9

• Primer binds to plasmid on the recombinant plasmid DNA strand.

Question 10

What happens at 60 degrees in DNA sequencing?

Answer 10

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

Question 11

How are terminator bases modified?

Answer 11

• Modified with coloured fluorescent tags

Question 12

Why does the process repeat?

Answer 12

To get different lengths of DNA fragments.

Question 13

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

Answer 13

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

Question 14

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

Answer 14

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

Question 15

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

Answer 15

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

Question 16

What is Pyrosequencing?
- think of mRNA
- complimenting

Answer 16

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

Question 17

What is a nebuliser?
- 🖥️

Answer 17

Used to mechanically cut DNA with into fragments

Question 18

What are activated nucleotides?
- what are the examples?

Answer 18

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

Question 19

What is Apyrase?
- Unincorporated A N

Answer 19

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

Question 20

1. (Pyrosequencing) what happens to the large piece of DNA?
   -what equipment used?

Answer 20

• large sequence of DNA is cut
• using nebuliser

Question 21

2. (Pyrosequencing) What happens after fragments are formed?

Answer 21

The DNA is degraded to a single strand (ssDNA)

Question 22

Define what ssDNA is?

Answer 22

Single strand DNA

Question 23

3. (Pyrosequencing) what happens after ssDNA is made?
   -SP

Answer 23

• sequencing primer is added to ssDNA.

Question 24

4. (Pyrosequencing) when sequencing primer is added what is the DNA incubated with? 6
   - poly.., ATP s (enzyme), luciferase, apyrase, APS, AN

Answer 24

• DNA polymerase
• ATP Sulfurylase
• Luciferase
• Apyrase
• Adenosine 5’ phoshosulfste (APS)
• One Activated Nucleotide

Question 25

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

Answer 25

• one activated nucleotide incorporates to complementary strand sequenced by template.

Question 26

6. (Pyrosequencing) what happens after nucleotide is added?

Answer 26

• the two extra phosphoryl are released as 2 phosphate (ppi)

Question 27

7. (Pyrosequencing) what happen when phosphate is released?
   -prescience of…
   - the enzyme …
   - converts the ..(before product) to ..( end product)

Answer 27

• In the presence of APS
• The enzyme ATP Sulfurylase
• Converts Phosphate to ATP

Question 28

8. (Pyrosequencing) what happened after we made ATP. -Luciferin
   - in the presence of..
   - the enzyme..
   - converts lu… to Oxy…

Answer 28

• in the presence of ATP
• the enzyme lusiferase
• converts lucifern to oxylucifern

Question 29

9. (Pyrosequencing) after oxyluceferin is made what then happens
   HINT - it creates.. (end result)

Answer 29

It creates photons of light

Question 30

10. (Prysequencing) what happens at the end of the process to get more photons of light

Answer 30

• Repeat the process with other activated nucleotides.

Question 31

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

Answer 31

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

Question 32

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

Answer 32

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

Question 33

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

Answer 33

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

Question 34

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

Answer 34

-Bones
-Teeth

Question 35

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✂️

Answer 35

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

Question 36

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

Answer 36

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