Lecture 12

Question 1

What is a vector?

Answer 1

A specifically designed plasmid or bacterial virus that will “carry” and amplify the gene of interest

Question 2

What are restriction enzymes?

Answer 2

Bacterial enzymes that cut the phosphodiester bond at specific DNA sequences called restriction sites. Another name for them is endonucleases. Some RE’s create sticky ends and some make blunt ends.

Question 3

How were restriction enzymes discovered?

Answer 3

It was found that certain bacteria were resistant to some phages. The bacterial enzymes restrict phage replication.

Question 4

How is the host bacterial DNA protected?

Answer 4

Methylation. RE’s won’t cut methylated DNA but they will cut phage DNA bc it is unmethylated.

Question 5

What is palindromic DNA?

Answer 5

A segment of DNA where both strands have the same nucleotide sequence but in antiparallel orientation
GAATTC
CTTAAG

Question 6

What is the mechanism by which EcoR1 works?

Answer 6

EcoR1 binds to double stranded DNA as a protein dimer. One subunit attaches to each strand and cleaves the phosphodiester bond lead to separation of fragments. EcoR1 creates sticky ends.

Question 7

What is a blunt end?

Answer 7

RE cuts at the same position on each of the two antiparallel strands. It offers more cloning possibilities because any DNA segment can be ligated to blunt ends. However, there are no H-bonds to hold new DNA segment like with sticky ends.

Question 8

What are sticky ends?

Answer 8

RE cuts so that it leaves a pair of ends that each ahve an identical 4 base pair single stranded end. It is easier to ligate to other DNA if ends are compatible. Cloning with sticky ends requires both the donor and vector DNAs to be digested with the sam RE so hybridization can occur.

Question 9

What are the two ways in which genes can be amplified?

Answer 9

Cloning (in vivo) and PCR (in vitro)

Question 10

What is PCR?

Answer 10

Polymerase Chain Reaction: amplifies selected regions of DNA in vitro. The three steps are 1) 95 degrees to denature DNA 2) 50-65 degrees for primers to anneal 3) 72 degrees elongation. These steps repeat about 33 times. Leads to billion fold amplification.

Question 11

What are 4 ingredients necessary for PCR?

Answer 11

primers, Taq polymerase, dNTPs, DNA, MgCl2

Question 12

What is cDNA? How is it created?

Answer 12

Complementary DNA: does not contain introns, only contains coding regions. It is created by reverse transcribing mRNA.

Question 13

What is agarose gel electrophoresis?

Answer 13

Technique used to separate linear DNA based on size. DNA is inserted into wells which are near the cathode end (neg charged) –> DNA migrates down toward anode because DNA is neg charged. Speed of migration depends on size. Smaller DNA segments migrate faster. Visualization of gel is done by ethidium bromide staining (fluoresces under UV).

Question 14

What is cloning?

Answer 14

Amplification achieved in vivo by enzymes in a living cell. A single recombinant vector enters a bacterial cell and is amplified by the same machinery that replicates the bacterial chromosome. The recombinant vector must have an origin of DNA replication recognized by host replication proteins. Cloning leads to many copies of each vector in each bacterial cell.

Question 15

Is PCR or cloning faster?

Answer 15

PCR, but it requires knowledge of the DNA sequence in order to create primers. Cloning is slower, but only requires knowledge of nearby restriction sites.

Question 16

How can sticky ends be blunted to make them compatible for a DNA segment in cloning?

Answer 16

The Klenow fragment of DNA pol I can uses 5’ to 3’ polymerase activity to fill in recessed 3’ end OR uses 3’ to 5’ exonuclease activity to trim 3’ overhang.

Question 17

How are the gene and vector joined together?

Answer 17

DNA Ligase. It is more efficient if sticky ends are used because it has the secondary support of H-bonds.

Question 18

What does the amplification of donor DNA inside a bacterial cell entail?

Answer 18

1) Choosing a cloning vector and introducing insert
2) Introducing recombinant DNA molecule inside bacterial cell
3) Recovering amplified recombinant molecules

Question 19

What is a plasmid vector?

Answer 19

A small circular extrachromosomal DNA which can accommodate inserts <10kb. Usually carries a gene for drug resistance and a gene to distinguish plasmids with and without DNA inserts. For example, a DNA insert may disrupt a gene in the plasmid.

Question 20

What is a bacteriophage vector?

Answer 20

A modified bacterial virus which can accommodate inserts up to 20kb. The phage genome is cut out by REs and discarded then replaced by inserts of donor DNA. Each phage contains recombinant DNA and viral genes needed to create new phage particles.

Question 21

What is a fosmid?

Answer 21

A hybrid plasmid derived from bacteriophage lambda and bacterial F plasmid DNA. It can accommodate inserts up to 45kb. Fosmids are packaged into lambda phage particles which are then injected into recipient bacterial cells with recombinant DNA. Fosmids replicate extrachromosomally.

Question 22

What is a bacterial artificial chromosome (BAC)?

Answer 22

Accommodates inserts up to 200kb. Bacterial F plasmid is cut and inserted with donor DNA.

Question 23

Which vectors use transformation to enter the bacterial cell?

Answer 23

Plasmids and BACs –> form bacterial colonies

Question 24

Which vectors use transduction to enter the bacterial cell?

Answer 24

Fosmids –> form bacterial colonies

Question 25

Which vectors use infection to enter the bacterial cell?

Answer 25

Bacteriophage –> form phage plaques

Question 26

How are amplified recombinant molecules recovered?

Answer 26

Bacteriophage: college phage lysate and isolate DNA
Plasmids/Fosmids/BACs: Bacteria is broken apart then DNA plasmid is separated from the much larger main bacterial chromosome by centrifugation, electrophoresis, etc to distinguish the chromosome from the plasmid by size or shape.

Question 27

What is transfection? injection? electroporation? projectiles?

Answer 27

transfection: use of viral vector
injection: using fine glass needles
electroporation: electric shock makes pores
projectiles: gold and tungsten beads coated with DNA

Question 28

What are some problems with cloning?

Answer 28

1) Introns in eukaryotic DNA. Remedy = use cDNA
2) Promoter/ribosome binding site function. Remedy = vector supplies expression signals appropriate for host cell.
3) Phosophorylation/Glycosylation and other post translational protein modifications. Remedy = Clone into eukaryotic cell (yeast)

Question 29

What is a probe?

Answer 29

Will recognize a specific nucleic acid sequence OR a specific protein. They can radioactively labeled or fluorescent labeled. They are single stranded DNA or RNA and must be complementary to desired sequence.

Question 30

What is a southern blot?

Answer 30

Used to identify particular DNA segment of interest.

Ex. Genomic DNA is digested by RE’s –> gel is a schmear –> a probe can identify one fragment in the schmear

Question 31

What is a northern blot?

Answer 31

Used to detect a specific RNA molecule form a mixture of RNAs fractitioned on a gel. RNA is first separated by gel electrophoresis then hybridized with probe. RNA is not digested by REs.

Question 32

What is a western blot?

Answer 32

Uses anitbodies are probes to detect proteins in a gel. A protein mixture is run on a gel, blotted, then bathed with antibody to reveal position of protein.

Question 33

What are DNA microarrays?

Answer 33

DNA molecules representing many (all) genes are fixed to slide or chip. mRNA from tissue used to make labeled cDNA. Labeled cDNA washed over chip. This quickly identifies which genes expressed in tissue.

Question 34

What is Sanger sequencing?

Answer 34

Interrupted DNA replication. A way to sequence DNA by “reading” the bases. Sanger sequencing requires primer, DNA pol, dNTPs, and ddNTPS (lack 3’ OH). Each ddNTP has a different fluorescent tag. A laser and detector help detect the colors and therefore provide the order bases. Shorter DNA is at the bottom of capillary tube.

Question 35

What is high throughput (Next Gen) sequencing?

Answer 35

1) A DNA template library of ssDNA molecules is constructed
2) DNA is amplified by PCR –> single DNA strands are immobilized on individual beads –> PCR so each bead contains many identical fragments –> each bead is deposited into wells
3) Each bead is sequenced by pyrosequencing
The pattern of light flashes = sequence of bases

Question 36

What is pyrosequencing?

Answer 36

DNA pol and primer is added into each well –> one dNTP is introduced at a time in specific order –> if dNTP is added to chain then PPi is released –> sulfurylase converts PPi to ATP –> luciferase makes burst of light –> camera records wells that release light.