A6 - Gene Tech
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1
4 ways to isolate DNA
Reverse transcriptase
Restriction endonuclease
PCR
Gene machine
Restriction endonuclease
PCR
Gene machine
2
Describe how reverse transcriptase isolates DNA
Enzyme extracted from retrovirus (eg. HIV)
Enzyme makes complementary DNA copy of RNA
Produces cDNA (single stranded, only exons)
Enzyme makes complementary DNA copy of RNA
Produces cDNA (single stranded, only exons)
3
What is a restriction endonuclease
Enzyme that hydrolysed genetic material at specific base sequences
4
What are the 2 types of cuts?
Sticky ends
Blunt ends
Blunt ends
5
What are sticky ends?
Exposed complementary bases
6
What are blunt ends?
7
What does DNA ligase do?
Sticks strands of DNA together
8
Describe the process of using a gene machine
Aa sequence input into computer
Computer works out DNA sequence
Computer instructs gene machine
Required DNA (only exons) sequence made
Computer works out DNA sequence
Computer instructs gene machine
Required DNA (only exons) sequence made
9
What does PCR stand for
Polymerase chain reaction
10
What does PCR do?
Makes lots of copies of DNA
11
What are the temp changes (°C) in PCR?
94
50-60
74
50-60
74
12
Describe process of PCR
Heat to 94
Separwtes DNA strands by breaking H bonds
Cool to 60
Primers attach
Heated to 74
Heat stable DNA polymerase joins free DNA nucleotides
Separwtes DNA strands by breaking H bonds
Cool to 60
Primers attach
Heated to 74
Heat stable DNA polymerase joins free DNA nucleotides
13
Define primer
Short single strand of DNA
One binds to start and one binds to end
One binds to start and one binds to end
14
Define in vivo cloning
DNA fragments added to DNA as it grows and divides, replicating the foreign DNA
15
Example of in vitro cloning
PCR
16
Define vector
DNA carrier used to transfer DNA into cells
17
Examples of vectors
Plasmids in bacteria
Virus
Virus
18
Name for type of DNA formed from vector DNA and inserted DNA fragment
Recombinant DNA
19
Definemarker gene
Gêne inserted into vector which gives organism specific characteristic that can be used to identify the organism or if the insertion of a gene was successful
20
3 advantages of in vivo cloning
Clones large DNA fragments
Produces proteins / MRNA from the DNA
Produces only exons
Produces proteins / MRNA from the DNA
Produces only exons
21
3 advantages of in vitro cloning
Quicker - millions produced within hours
Only replicates target DNA (isolation not needed)
Can copy partly broken down DNA
Only replicates target DNA (isolation not needed)
Can copy partly broken down DNA
22
In gel electrophoresis, what type of DNA fragment moves the furthest?
Short
23
Which end to DNA fragments move towards in gel electrophoresis and why!
+ive
DNA is slightly - ive
DNA is slightly - ive
24
Define gene probe
Small section of single stranded DNA complementary to DNA fragment that needs to be located
Radioactivelly labelled
Radioactivelly labelled