7 Genes and Genomes

Question 1

How do geneticists replicate - measure - store - change DNA ?

Answer 1

replicate = PCR
measure = Gel electrophoresis
store = vectore libraries
change = mutagenises/CRISPR

Question 2

how do geneticists determine DNA sequence

Answer 2

Sangar or NExt gen sequencing

Question 3

How do geneticists determine when and where DNA is used

Answer 3

qPCR and in situ hybridization

Question 4

what are some facts about PCR

Answer 4

○ Done inside test tubes (invitro)
○ Amplifies DNA fragments
○ Requires primers

Question 5

what are some uses of PCR

Answer 5

○ DNA fingerprinting
§ Forensics
§ Paternity testing
§ Prenatal screening
○ Mutation detection
○ Detect specific pieces of DNA

Question 6

what are PCR requirements

Answer 6

○ Template DNA-from cells
○ 2 specific short Primers
§ Need forward and reverse primers
○ dNTPs
§ to make more DNA
○ Buffer
§ make environment ideal for enzymes
○ DNA polymerase
§ Synthesize DNA
§ Taq polymerase specifically (resistant to high temp)

Question 7

what are the steps to PCR

Answer 7

○ denaturation
§ Heat to separate DNA strand (95 deg)
○ annealing
§ Cool so primers bind (60 deg)
○ extension
§ Heat up to taq polymerase ideal temp (72 deg)
○ Taq synthesizes DNA (step is short bc we don’t want to synthesize the whole strand)

Question 8

what are intercalators in measuring DNA

Answer 8

§ Bind in DNA
§ They are fluorescent so we can see the DNA (it glows)

Question 9

what are vectors

Answer 9

○ Small, transferrable, and replicable DNA molecs

Question 10

what are 2 types of vectors

Answer 10

§ Plasmids (bacteria)
□ Circular DNA
□ Vary in size
§ Phage (virus)
□ Moonlander type shape
□ Use their genome to replicate our DNA we snuck into it

Question 11

what are some traits of gene cloning

Answer 11

○ In vivo (in life) system
○ Insert into viable host molecule
§ This is our vector
○ Can be extracted and purified

Question 12

what are traits of engineered plasmids

Answer 12

○ starts at Ori
§ Origin of replication
§ How plasmid replicates itself
§ Where polymerase binds
○ AmpR (ampicillin resistant)
§ Selective marker
§ Prevents bacteria from dying from ampicillin
○ Has polylinker site
§ Where we put our DNA insert

Question 13

how do we fuse DNA into recombinant molecs

Answer 13

○Use Restriction enzymes
§ Type of endonuclease
§ Make site specific cuts in DNA
□ Cuts at the restriction site
□ Cuts double stranded DNA

Question 14

how does Ligation of an insert into a vector work

Answer 14

• circular plasmid (vector) opened up with a restriction digestion
• insert placed into opened DNA with restriction enzymes to make matching end
• ligase + plasmid backbone inserted

Question 15

how do we get our ligated DNA product (circular plasmid DNA we changed) in the cell

Answer 15

• heat shock or electroporation (electric shock) so the membrane will allow our vector in

Question 16

what is the cloning rationale (steps)

Answer 16

1) Extract and isolate gene from cell
a. PCR
b. Gel
c. Restriction enzyme
2) Ligation
3) Clone into the vector model
a. Viral or plasmid
4) Transformation
5) Introduce vector into host cell
a. Bacteria replicates
6) extraction
a. Retrieve gene from host

Question 17

TF What needs to be in both the vector and the insert

Answer 17

restriction enzymes

Question 18

what is the pahge life cycle that we want in regards to cloning

Answer 18

The lytic cycle

Question 19

what is the lytic cycle in a phage lifecycle

Answer 19

After out phage with out DNA is inserted into the cell
- our DNA will replicate and make many viral chromosomes
- our phages will be created in the cell
- at the end there is no circular DNA in the cell and our phages will burst from the cell wall

Question 20

why do we not care about the lysogenic cycle

Answer 20

because we want them right away and the lysogenic cycle takes time

Question 21

how do we find the bacteria that we want from the cells

Answer 21

Kill everything else
○ 2 steps to this process
§ Select for plasmid
□ Ensure that only the bacteria with the plasmid will survive (Antibiotic resistance)
§ Selection for insert
□ Identify bacteria with recombinant plasmids
□ Blue/white selection

Question 22

why does the bacteria we want not die when we add the antibiotics

Answer 22

our bacteria has AmpR (ampicillin resistance)

Question 23

what does the LacZ gene produce

Answer 23

Beta-galactosidase

Question 24

TF LacZ turns X-Gal Red

Answer 24

F, turns it blue

Question 25

TF if the cell is blue then its our desired clone

Answer 25

F,if its white its our desired clone

Question 26

if LacZ is broken what colour is it

Answer 26

white

Question 27

Most live cells are ___

Answer 27

blue

Question 28

what is broken on the vector (DNA) in order for our insert to be added

Answer 28

the polylinker site

Question 29

what needs to be in our cell in order for it to end up being a viable clone

Answer 29

• 1 AmpR gene
• Our inserted genes
• polylinker on either side of our inserted gene

Question 30

what do we need to avoid when inserting our DNA into the plasmid

Answer 30

cleaving inside the insert or outside the polylinker (dont break other things)

Question 31

when looking at which phage to select the one that we want (full of virus is) _______ and the one we dont want (no virus) is ______ and one with unwanted virus is ______

Answer 31

• a solid circle
• a live bacteria
• a hollow circle

Question 32

what is a transgenic organism

Answer 32

○ organism that contains transgene

Question 33

what can transgenes be used for

Answer 33

○ Biotechnology
○ Research

Question 34

why is yeast a good transgene

Answer 34

• Eukaryotic
• Unicellular
• Grow quickly
• Can be Haploid and diploid

Question 35

what is a transposable element

Answer 35

• DNA sequence that can move itself in the genome

Question 36

what are 2 types of transposable elements

Answer 36

○ DNA transposons
○ Retrotransposons

Question 37

TF A huge portion of our genome is transposable elements

Answer 37

T

Question 38

what are C.elegans

Answer 38

• Microscopic nematode (worm)
• Have exactly 959 cells
• Transgenes don’t integrate into the genome so they create Extrachromosomal arrays

Question 39

what is an extrachromosomal array

Answer 39

as eggs develop they hang on to it and the eggs act as if it was an extra chromo and will grow up transgenic

Question 40

what is the way to get transgenes into vertebrates

Answer 40

transgenes are added by pulling egg out and gene is inserted by needle into the egg

Question 41

What can CRISPR do ?

Answer 41

inactivate gene of interest
add genes of interest
modify gene of interest
modify gene regulation

Question 42

how does crispr inactivate genes of interest

Answer 42

a. Relies on NHEJ
b. Use 2 cut sites

Question 43

how does crispr add genes of interest

Answer 43

a. Relies on HR
b. Use 2 cut sites (increase chances of full repair)
c. Added DNA contains homologous flanking regions and gene of interest
d. Take gene out, add our gene into it and then put both back

Question 44

how does cripsr modify genes of interest

Answer 44

a. Relies on HR
b. cuts 2x
c. Added DNA contains flanking regions

Question 45

how does crispr modify gene regulation

Answer 45

a. Cas9 modified as gene regulator
b. Binds to promoter region
i. Can turn on/off specific genes as a transcription factor
ii. Causes permanently active Ins1

Question 46

what is the main protein used in CRISPR

Answer 46

Cas9

Question 47

what is the job of the Cas9 Protein

Answer 47

to cut DNA (causes dbl stranded break)

Question 48

the Cas9 protein is binded to what

Answer 48

guide RNA

Question 49

what happens in crispr after Cas9 has made a dbl stranded break in the DNA

Answer 49

repaired by cell thru NHEJ or HR
NHEJ - errors likely
HR - adds homologous DNA (fills genome with what we give it)