Tools of Molecular Genetics Flipped Classroom

Question 1

cDNA

Answer 1

made as copy of RNA, no introns, used to determine AA sequence or make large quantities of protein by cloning

Question 2

Dideoxyseq

Answer 2

Utilize polyermase and chain terminating nucs

READ FROM THE BOTTOM UP!!!! (longer goes further)

Question 3

Microarray

Answer 3

large # of short DNA molecules that are immobilized
Each DNA fragment acts as probe for certain gene
Have two mRNA-whicheever lights up more has more expression of the gene

Question 4

Hybridization

Answer 4

have 2 complementary nucleic acid strands to form a duplex-used in detecting specific nucleotide sequences

Question 5

Transfection

Answer 5

Euk cells take up DNA from surroundings

Question 6

Transformation

Answer 6

Prok cells take up DNA from surroundings

Question 7

Gene knockdown

Answer 7

Downregulate expression using RNAi

Question 8

Palindromic

Answer 8

Two fold sum-looks the same twice in 360 degree turn

Question 9

DNA electrophoreresis

Answer 9

Runs from + to -
Smaller move faster
May have to denature RNA
Ethidium bromide/radioisotope stain

Question 10

Hybridization conditions

Answer 10

Higher-lower probablty of hybridization
-use formamide (denature) or more heat, shorter probe
Lower-higher probability of hybridization
-Use less formaminde and less heat, longer probe

Question 11

RFLP

Answer 11

detect by running gel electrophoresis

Question 12

3 blotting techs

Answer 12

North-RNA, South-DNA, West-Protein

Question 13

Steps of blotting

Answer 13

1. Extract whatever from where and purify
2. Place into gel and run
3. Transfer to nitrocellulose or nylon paper
4. Add radioactive probe
5. Wash and xray

Question 14

Allele Specific Oligos

Answer 14

Match nucleotide seq flanking substitution, typically use wt and mutant copies of gene as probe, see if have mutant or wt copy in cell
Keep in mind each cell has two copies of protein, heaterzygotes will always show up as positive

Question 15

PCR steps

Answer 15

1. heat to separate strands
2. Cool with excess of primers
3. incubate with DNA poly and 4 deoxy nucs

Question 16

Allele specific PCR

Answer 16

want to create a lot of specific allele

Must have near perfect 3’ end binding

Question 17

RT PCR steps

Answer 17

1. isolate mRNA
2. create cDNA with revtranscriptase
3. Primers

Question 18

Automated DNA seq

Answer 18

PCR makes fragments that are separated by capillary gel electrophoresis-read color as go down tube

Question 19

Construct cDNA library steps (and how to tell which DNA has what gene)

Answer 19

1. Cleave DNA with endonuclease
2. Use ligase to put fragments into plasmids
3. Transfect into proks
   +palce paper on proks, take off paper and add probe, take off and see what lights up under autoradiogrpahy

Question 20

Serial cloning steps

Answer 20

1. endonuc a plasmid
2. ligate dna
3. repeat as desired
   can engineer hybrid proteins with fluorescent marker to see what took-or use reporter gene

Question 21

Make large amounts of protein

Answer 21

Place next to strong promoter

Question 22

Epitope tagging

Answer 22

epitope for specific antibody is fused to target protein

• useful for monitoring protein expression/localization/topological dynamics/interations
• can use to do column separation

Question 23

Knokcout and replacement

Answer 23

no gene active and only mutant gene active