Lec 6-1

Question 1

Recombinant DNA techniques

Answer 1

Gene cloning

Restriction enzymes

Visualizing DNA

Question 2

Cloning genes

Answer 2

Using plasmid vectors (artificial DNA engineered to carry custom DNA sequences and genes)

Transformation of host cells with plasmids

Screening cells for recombinant plasmids

Question 3

Gene cloning

Plasmids and bacterial transformation

Answer 3

We co-opt bacteria’s transformation ability to incorporate plasmid DNA for gene cloning

We can artificially make bacteria competent

Question 4

Competent

Answer 4

Able to take up foreign walls by making the cell walls porous (can be done through heat shock)

Question 5

Transformant

Answer 5

Bacterial cell that has taken in the plasmid

Question 6

Replication can happen

Answer 6

Independent of bacterial cell genome using an origin of replication encoded in a DNA sequence in the plasmid

Question 7

An ideal cloning vector has

Answer 7

An origin of replication

One or more selectable markers

Recognition sites for one or more restriction enzymes

Question 8

Advantages of plasmids and bacterial transformation

Answer 8

Easy expansion- Bacterial cells can be quickly and cheaply expanded

Indefinite storage- Can be frozen and stored with little to no effect on bacterial cell health or degradation of DNA

Circular DNA is very stable

Question 9

How do we obtain/purify only transformants

Answer 9

Through one or more selectable markers

Question 10

Selectable markers

Answer 10

Genes that encode a marker, typically visual or growth selective

Allows only transformants to grow into colonies

A single colony is picked and cultured into large isogenic (same genotype) bacterial cultures

Plasmid DNA can be purified from these cultures

Question 11

Restriction enzymes

Answer 11

DNA endonuclease enzymes

Discovered in bacteria

Defense mechanism against invading viruses

Functions as an innate immune system

Question 12

Restriction enzyme have 3 ways cutting strands

Answer 12

5’ overhang
3’ overhang
blunt end

Question 13

Restriction enzymes and DNA ligases

Answer 13

Two DNA sources cut with same enzyme and mixed

Overhang ends will hybridize due to the correct homology

Question 14

Brightness of gel in ladders tells us

Answer 14

Size and how much is present in each ladder

Question 15

Challenges of molecular genetics

Answer 15

DNA is extremely thin

Finding 1 gene specifically is like finding a needle in a haystack

Question 16

Recombinant DNA capabilities

Answer 16

Locate a gene/DNA sequence

Remove/Copy DNA sequences

Visualize DNA

Store new DNA sequences

Question 17

Recombinant DNA means

Answer 17

Combining DNA from different sources

Question 18

Plasmids

Answer 18

Small circular double stranded DNA that can naturally exist in bacterial cells along side the bacterial genome

Question 19

T4 DNA ligase is from

Answer 19

Bacteria

Question 20

T4 DNA ligase

Answer 20

Covalently seals (ligates) nicks in sugar-phosphate bonds on two ends

Thus, creates stable double stranded piece of DNA

Question 21

Restriction enzymes and DNA ligases allow us to

Answer 21

Cut, insert, and reorganize different DNA sources

Thus— recombinant DNA

Question 22

Cloning vectors

Recognition sites for one or more restriction enzymes

Answer 22

Can have many different restriction sites

Question 23

Restriction enzymes are named such that

Answer 23

the first portion in italics represents the species of bacteria they were discovered in

Question 24

Visualizing DNA through gel electrophoresis

Answer 24

Gels are made that consist of a matrix with tiny holes

One side of the gel has small wells where DNA can be loaded

Question 25

visualizing DNA through gel electrophoresis

Answer 25

Gels covered in ionic buffer and an electrical field is applied Acids are negatively charged in this buffer DNA molecules will slowly move toward the other end of the gel (positive end)

Question 26

Gel electrophoresis Larger DNA fragments move

Answer 26

Slower through the matrix, thus run slower than small DNA fragments

Question 27

To visualize DNA in the gel Gel electrophoresis

Answer 27

DNA is stained with something that binds nucleic acids, such as Ethidium Bromide This can be imaged with a camera and an appropriate light source Certain DNA bands can be cut out and purified

Question 28

Polymerase chain reaction capabilities

Answer 28

Locate a gene/DNA sequence Remove/Copy DNA sequence

Question 29

Polymerase Chain Reaction (PCR)

Answer 29

Allows us to copy and then multiply a specific DNA sequence in an efficient matter

Question 30

PCR is developed by

Answer 30

Kary Mullis

Question 31

PCR is inspired by

Answer 31

How cells replicate DNA