Recombinant DNA Technology

Question 1

basic procedure for recombinant DNA technology

Answer 1

-generating specific DNA fragments using restriction enzymes
-joining these fragments with a vector
-transferring recombinant DNA to host cell to produce many copies that can be recovered from host cell

Question 2

what is a clone

Answer 2

recovered copies of a recombinant DNA molecule

Question 3

2 important tools used to construct and amplify DNA molecules

Answer 3

restriction enzymes
cloning vectors

Question 4

what do restriction enzymes bind to

Answer 4

restriction sites

Question 5

what is the symmetry that recognition sequences exhibit?
how long can they be?

Answer 5

palindromes
-most 4-6 nucleotides(few contain 8+)

Question 6

sticky vs blunt end?

Answer 6

Sticky: single stranded segments produced by some restriction enzyme cuts(can base pair with complementary sequences)

Blunt: no single stranded overhangs

Question 7

restriction site for HindIII, EcoRI, BamHI, and AluI?
what end(blunt or sticky) is formed?

Answer 7

HindIII: A/AGCTT, sticky
EcoRI: G/AATTC, sticky
BamHI: G/GATCC, sticky
AluI: AG/CT, blunt

Question 8

procedure for creating recombinant DNA with restriction enzymes

Answer 8

-cleave DNA from 2 sources with the same restriction enzyme
-mix fragments
-incubate with DNA ligase to link strands covalently

Question 9

what is a restriction map?
how can they be used?

Answer 9

maps of DNA sequences made from restriction experiments
-provide info for subcloning

Question 10

what were the first vectors developed

Answer 10

genetically modified bacterial plasmids

Question 11

what is a plasmid

Answer 11

extrachromosomal double stranded DNA molecule that replicates independently from chromosomes within bacterial cells

Question 12

how can selectable markers identify recombinant DNA?
what are examples of the genes used?

Answer 12

-genes that provide resistance to antibiotics
-ampicillin and lacZ

Question 13

how is transformation achieved

Answer 13

-using calcium ions and heat shock to pulse DNA through cells
-electroporation: intense pulse of electricity to move DNA through cells

Question 14

what is the result of blue-white screening mechanism?
what components does it use?

Answer 14

identifies recombinant bacteria
-lacZ encode B-galactosidase
-agar plate
-X gal

Question 15

Blue v. white colonies (recombinant or non recombinant)

Answer 15

Blue: non-recombinant
White: recombinant

Question 16

what are other types of vectors

Answer 16

-phage vectors
-expression vectors
-eukaryotic expression vectors
-bacterial artificial chromosome(BACs)

Question 16

what is a BAC/YAC?
what is the insert size capacity of a BAC?

Answer 16

BAC: bacterial artificial chromosome, 100-200 kb
YAC: yeast artificial chromosome

they are vectors used to clone large fragments of DNA

Question 17

what alongside soil bacterium can be used for introducing genes in plants

Answer 17

Ti plasmid

Question 18

when are eukaryotic expression vectors used

Answer 18

yeast or tissue culture where bacteria cannot produce functional products of transgene

Question 19

DNA v. genomic v. cDNA libraries

Answer 19

DNA: cloned fragments of DNA from a single source

Genomic: contains many overlapping fragments of the genome

cDNA: derived by mRNA, complimentary DNA copies

Question 20

T/F genomic libraries contain all of the DNA

Answer 20

TRUE. contain at least one copy of every DNA sequence in an organisms chromosome

Question 21

what is a probe

Answer 21

DNA or RNA sequence complementary to target gene of sequence being identified

Question 22

steps in PCR with temps

Answer 22

-denaturation(92-95 C)
-anneal primers(45-65 C)
-extent primers(65-75 C)

Question 23

RT-PCR v. qPCR

Answer 23

RT-PCR: reverse transcription, studies gene expression

qPCR: quantitative real time, quantify amplification reactions in real time

Question 24

qPCR can evaluate gene expression when correlated to what output

Answer 24

fluorescence(RFU)

Question 25

what is gene targeting

Answer 25

target specific allele, locus, or sequence to learn its function on gene of interest

Question 26

what is gene knowckout

Answer 26

loss of function mutation -eliminate gene/genes to see what happens

Question 27

T/F insulin was the first human genes to be expressed in E. coli

Answer 27

TRUE

Question 28

the AA sequence of insulin was determined in the ______

Answer 28

1950s

Question 29

what cells did knockout mice use to be injected into a blastocyst

Answer 29

embryonic stem cells

Question 30

transgenic model example? what was it trying to achieve? what was the end result?

Answer 30

loss-of-function cystic fibrosis -produce offspring that were heterozygous for the transgene

Question 31

best approach for genome editing

Answer 31

CRISPR-cas9

Question 32

T/F germinal gene therapy has been performed in humans

Answer 32

FALSE

Question 33

T/F Crips-cas9 used sgRNA and dsRNA break repair mechanisms

Answer 33

TRUE

Question 34

gene editing with CRISPR-cas9 has been used to delete mutant exon 23 from a mouse model of DMD loss of function alleles in what gene?

Answer 34

dystrophin gene

Question 35

who was the first cloned cat

Answer 35

CC(copy cat)

Question 36

what was the first institution to clone 3 species? what cloned species were discussed in class?

Answer 36

A&M cloned first horses, pigs, goats, and deer were discussed in class