Lecture 17

Question 1

In what aspects has recombinant DNA technology contributed to our lives?

Answer 1

• 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

Question 2

Restriction endonuclease

Answer 2

• 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

Question 3

Gel electrophoresis

Answer 3

• 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

Question 4

What affects do changing the temperature on DNA have

Answer 4

• 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

Question 5

Compare southern blotting with northern blotting

Answer 5

• 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

Question 6

How to create recombinant DNA

Answer 6

• 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)

Question 7

DNA cloning:

Answer 7

• Producing many identical of copies of a DNA sequence

Question 8

Recombinant DNA:

Answer 8

a DNA molecule that contains DNA from multiple sources

Question 9

Plasmid:

Answer 9

small circular DNA molecule often used in bacteria (and in the lab)

Question 10

How are plasmids introduced into bacteria?

Answer 10

• 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

Question 11

what is a DNA library

Answer 11

• a collection of DNA clones

Question 12

What are the different types of libraries

Answer 12

1. 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
2. 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

Question 13

How to create a genomic library

Answer 13

1. DNA is digested using RE or DNA shearing
2. DNA fragments are cloned into plasmids
3. Plasmids are introduced into E. coli as hosts

Question 14

How to create cDNA library

Answer 14

1. total RNA is extracted from an organism, then isolate mRNA
2. complementary DNA is prepared using a reverse transcriptase using a polyT primer complementary to polyA tail of mRNA
3. the cDNA is inserted into a vector (plasmid) and clone to produce a cDNA library
4. cDNA can be inserted into a vector
5. vector is introduced to bacteria

Question 15

What is a polymerase chain reaction (PCR)

Answer 15

• 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

Question 16

What are the 3 steps to PCR

Answer 16

1. heat to separate DNA
2. cool and anneal primers that flank sequence of interest
3. allow DNA polymerase to extend from the primers
   Notes: this is the first cycle of amplification, steps 1 through 3 are repeated 30 to 35
   - amplification = 2^n where n = # of cycles

Question 17

Why do we need thermostable DNA polymerase in PCR

Answer 17

• if we didn’t the DNA polymerase would denature when it was heated, meaning we would need to add it fresh after each cycle

Question 18

When PCR is used

Answer 18

• PCR is used while generating DNA libraries to amplify segments to be cloned (clone genes)
• used detect small amount of RNA or DNA from pathogen
• used to amplify STRs to be used during DNA fingerprinting

Question 19

Sanger sequencing

Answer 19

• makes use of dideoxyribonucleoside triphosphates (ddNTPs)
• ddNTPs have a 3’H end instead of a 3’OH which makes DNA polymerase impossible

Question 20

the process of sanger sequencing

Answer 20

• the DNA of an unknown sequence is put into 4 different reaction tubes
• tube contains ATP, GTP, CTP, TTP, DNA polymerase and Mg2+
• each tube contains different ddNTPs at low concentration
• the ddNTPs terminate synthesis when incorporated but termination occurs at different places depending on where it was incorporated
• the different sized fragments are resolved on a gel and the sequence of the template is determined

Question 21

automated sanger sequencing

Answer 21

• uses same principle but instead of using radioactive label each ddNTP is linked to a different fluorescent molecules giving all fragments terminating in that nucleotide a different color