Lecture 17 Flashcards
1
Q
In what aspects has recombinant DNA technology contributed to our lives?
A
- The ability to manipulate DNA has advanced our understanding of molecular biology
- It has contributed to many aspects in our lives such as:
- Health and medicine
- Industrial advancements
- Forensics
- Environment
2
Q
Restriction endonuclease
A
- Are “DNA scissors” that cleave DNA at specific sequence
- Most restriction recognition sequences are 4-6 base pairs long (some are 8) that are palindromic
- Cuts can be blunt or leave a sticky overhang
HaeIII: GGCC
EcoRI: GAATTC
HindIII: AAGCTT
3
Q
Gel electrophoresis
A
- Can be used to separated DNA of different sizes
- DNA is loaded into wells of agarose gel and an electric field is applied
- DNA migrates towards the positive electrode; smaller DNA migrates through the gel matrix faster than larger fragments
- DNA can be visualized by staining with a dye or using DNA with a radioactive label
4
Q
What affects do changing the temperature on DNA have
A
- Increasing temperature can denature DNA to release single strands because hydrogen bonds between nucleotides break
- Decreasing the temperature can cause the strands to renature
- This is hybridization: the process of DNA renaturation
5
Q
Compare southern blotting with northern blotting
A
- Southern blotting involves the transfer of DNA to a membrane and hybridization with a labeled probe
- Northern blotting is the same process but with RNA
6
Q
How to create recombinant DNA
A
- Insert our DNA in our plasmid by cutting both with a Sa/I
- Step 1: Cut the DNA of interest with Sa/I
- Step 2: Cut the plasmid vector with Sa/I
- Step 3: insert DNA into the plasmid and ligate the two together
- Step 4: reseal nicks with DNA ligase + ATP (plasmid now contains DNA of interest)
7
Q
DNA cloning:
A
- Producing many identical of copies of a DNA sequence
8
Q
Recombinant DNA:
A
a DNA molecule that contains DNA from multiple sources
9
Q
Plasmid:
A
small circular DNA molecule often used in bacteria (and in the lab)
10
Q
How are plasmids introduced into bacteria?
A
- Through transformation
- Once bacteria takes up plasmid, when they replicate, the plasmid will also replicate so you will be left with a lot of copies
11
Q
what is a DNA library
A
- a collection of DNA clones
12
Q
What are the different types of libraries
A
- Genomic library:
- representative of all of the genomic sequence of an organism
- includes both coding and non-coding DNA
- library will essentially be the same regardless of the cell type used for prep - cDNA library
- contains only genes that are transcribed into mRNA
- clones contain regions only of the genome that have been transcribed to mRNA
- gene expression varies from one cell to another, therefore a distinct cDNA library is obtained fro each cell type used for the preparation
13
Q
How to create a genomic library
A
- DNA is digested using RE or DNA shearing
- DNA fragments are cloned into plasmids
- Plasmids are introduced into E. coli as hosts
14
Q
How to create cDNA library
A
- total RNA is extracted from an organism, then isolate mRNA
- complementary DNA is prepared using a reverse transcriptase using a polyT primer complementary to polyA tail of mRNA
- the cDNA is inserted into a vector (plasmid) and clone to produce a cDNA library
- cDNA can be inserted into a vector
- vector is introduced to bacteria
15
Q
What is a polymerase chain reaction (PCR)
A
- a very powerful technique that allows you to create billions of copies of nucleic acid
- does not require a living cell
- extremely sensitive
- very fast and easy to do
