Topic 7

Question 1

What is PCR?

Answer 1

amplify DNA molecules using primers, and heating and cooling cycle

Question 2

What is gene cloning?

Answer 2

in vivo system to build new combination of genes

Question 3

How do restriction endonucleases work?

Answer 3

recognize a specific DNA sequence and cleave DNA in predictable site in the DNA

Question 4

How do you purify DNA fragments?

Answer 4

Agarose gel electrophoresis

Question 5

Where are larger fragments in the gel?

Answer 5

closer to the top

Question 6

What makes the DNA fragments fluoresce?

Answer 6

ethidium bromide

Question 7

What are vectors?

Answer 7

small, transferrable, and replicable DNA molecules that can replicate in a live host

Question 8

What are the two main types of vectors?

Answer 8

phages and plasmid

Question 9

Why are plasmids useful for cloning?

Answer 9

replicate autonomously, AMR genes for selectable markers

Question 10

What are the three necessary components for a plasmid?

Answer 10

1. ori
2. selective marker
3. multiple RE sites for insertion

Question 11

What are some things you have to keep in mind for choosing restriction sites in insert and plasmid?

Answer 11

be sure not to cut in insert or in functional areas of plasmid (i.e. replication, resistance)

Question 12

What are the two most common selection markers for plasmids?

Answer 12

Ampicillin resistance and LacZ’ gene

Question 13

What are one of the issues with ampicillin resistance selection?

Answer 13

don’t know if live cells have empty plasmid or plasmid with genes

Question 14

What is B-galatosidase and why is it important in cloning?

Answer 14

involved in lactose metabolism and recognizes analogs and will turn blue when metabolized

Question 15

What is a-complementation?

Answer 15

When plasmid LacZ and LacZ Omega produce subunits to reconstitute a functional B-Gal enzyme

Question 16

Do blue or white colonies indicate successful cloning into a cell and why?

Answer 16

white because it inserts into LacZ’ frame meaning no complementation

Question 17

How do you ensure directional cloning?

Answer 17

cut with two REs

Question 18

What portion of the bacterial chromosome is not necessary for cloning purposes?

Answer 18

lysogeny part

Question 19

What are some downsides to using bacteriophages for cloning?

Answer 19

foreign DNA must be 10-15kb

Question 20

How do you select for cloned colonies with bacteriophages?

Answer 20

plaque formation-clear bacteria

Question 21

How does Sanger sequencing work?

Answer 21

chain terminators are added so that everytime there is a complementary base pair it causes premature stopping

Question 22

How does automated Sanger Sequencing work?

Answer 22

ddNTPs are labeled with fluorophores so you can detect base pairs in a single reaction

Question 23

What are the drawbacks to Sanger Sequencing?

Answer 23

slow, expensive

Question 24

What are the three steps of Second gen NGS (Illumina)?

Answer 24

1. Sequencing library prep
2. Flow cell prep
   3.Parallel Sequencing

Question 25

How is the library prepared for Illumina sequencing?

Answer 25

gDNA is fragmented and adaptors are added to each end

Question 26

How do the flow cells work?

Answer 26

complementary sequences to the adaptors used are attached to flow cells, DNA binds to adaptor and comp. oligo, bend over (bridge) , PCR amplifies products

Question 27

How does parallel sequencing work?

Answer 27

each dNTP is labeled a different colour and when it is added it will emit a colour and be read by a scanner

Question 28

How can computers tell if the sequence is a protein coding gene?

Answer 28

identify by open reading frames

Question 29

How can we know whether expressed regions have functions?

Answer 29

look at conserved regions between animals and such

Question 30

What are Ultra conserved regions?

Answer 30

sequences in non coding regions that may play a role in regulating gene expression

Question 31

What are homologs?

Answer 31

genes that share a common ancestor and display significant sequences conservation

Question 32

What are orthologs?

Answer 32

homologous genes that are located in the same genetic locus in two closely related species

Question 33

What are paralogs?

Answer 33

genes in a genome that arose by duplication of a common ancestor gene

Question 34

If 99% of genes contain a corresponding mouse homolog, why don't we look like mice?

Answer 34

genes have appeared and have been lost over evolution

Question 35

What are pseudogenes?

Answer 35

genes that had function in the ancestral genome but have since accumulated mutations

Question 36

Why are we different than apes?

Answer 36

changes in regulatory sequences (cis elements)

Question 37

Why don't men have sensory whiskers like cats?

Answer 37

lost cis elements in androgen receptor