DNA tech

Question 1

What is a DNA library?

Answer 1

A collection of subdivided portions of a genome created by cloning DNA fragments into vectors.

Question 2

How is a genomic DNA library commonly created?

Answer 2

By partial digestion of genomic DNA with restriction endonucleases followed by cloning into vectors.

Question 3

What is the result of transforming cells with recombinant DNA fragments?

Answer 3

A collection of transformed cells called a library.

Question 4

What is the difference between DNA cloning and molecular hybridization?

Answer 4

DNA cloning amplifies the desired fragment; molecular hybridization detects it without amplification.

Question 5

Why is partial digestion used when making genomic libraries?

Answer 5

To generate overlapping DNA fragments of suitable size.

Question 6

What is size fractionation used for in library construction?

Answer 6

To collect DNA fragments of a desired target size.

Question 7

What is coligation?

Answer 7

Ligation of two unrelated DNA fragments into one recombinant molecule.

Question 8

What is transformation in cloning?

Answer 8

Uptake of recombinant DNA by host cells.

Question 9

Why are colonies picked after transformation?

Answer 9

To ensure clone homogeneity during amplification.

Question 10

What determines the number of clones that must be screened?

Answer 10

The genome size of the organism.

Question 11

What is a probe in hybridization screening?

Answer 11

A labeled DNA or RNA fragment (≥100 bp) used to detect complementary sequences.

Question 12

What level of sequence match is ideal for probe hybridization?

Answer 12

Greater than 80%.

Question 13

What is functional complementation?

Answer 13

Restoration of a lost function in a mutant strain using cloned DNA.

Question 14

What is the size limitation of PCR for genomic cloning?

Answer 14

Up to ~5 kb

Question 15

What are the three basic PCR steps?

Answer 15

Denaturation

Question 16

Which enzyme is used in PCR extension?

Answer 16

Taq DNA polymerase I.

Question 17

How many cycles are typically performed in PCR?

Answer 17

30–40 cycles.

Question 18

What is the amplification level of PCR?

Answer 18

Approximately 10^8-fold.

Question 19

What are SNPs?

Answer 19

Single nucleotide polymorphisms—single base pair variations in genomic DNA.

Question 20

How frequent are SNPs?

Answer 20

Approximately 1 per kb.

Question 21

What is RFLP?

Answer 21

Restriction Fragment Length Polymorphism caused by base changes altering restriction enzyme sensitivity.

Question 22

Applications of RFLP?

Answer 22

QTL analysis

Question 23

What mutation causes muscle hypertrophy in Texel sheep?

Answer 23

G→A mutation in myostatin gene creating a miRNA target.

Question 24

What is currently used for DNA identity profiling?

Answer 24

PCR amplification of short tandem repeats (STRs).

Question 25

What type of RNA is isolated to construct cDNA libraries?

Answer 25

Poly(A)+ mRNA.

Question 26

Which enzyme synthesizes cDNA?

Answer 26

Reverse transcriptase.

Question 27

What is the function of RNase H in cDNA synthesis?

Answer 27

Degrades RNA in DNA:RNA hybrids.

Question 28

What does S1 nuclease degrade?

Answer 28

Single-stranded nucleic acids.

Question 29

Purpose of enriching for full-length cDNA?

Answer 29

To ensure complete gene representation in expression libraries.

Question 30

Why is biotin used in full-length cDNA enrichment?

Answer 30

To capture full-length cDNA attached to biotinylated mRNA.

Question 31

What are the purposes of gene transfer into animal cells?

Answer 31

Study gene function

Question 32

What is transduction?

Answer 32

Viral delivery of DNA into cells.

Question 33

What is transfection?

Answer 33

Chemical or physical introduction of DNA into cells.

Question 34

Name chemical transfection methods.

Answer 34

Ca-phosphate and polycationic compounds.

Question 35

Name physical transfection methods.

Answer 35

Microinjection

Question 36

What is chemical transformation in bacteria?

Answer 36

CaCl2 treatment followed by heat shock.

Question 37

How does electroporation work?

Answer 37

High-voltage pulses create membrane openings allowing DNA entry.

Question 38

Why is electroporation preferred for large plasmids?

Answer 38

It is 10–100 times more efficient.

Question 39

What is gene targeting?

Answer 39

Altering gene function via homologous recombination.

Question 40

What are insertion vectors?

Answer 40

Targeting vectors with 5–20× higher frequency than replacement vectors.

Question 41

What are replacement vectors?

Answer 41

Vectors that replace a genomic segment via homologous recombination.

Question 42

Name dominant selectable markers.

Answer 42

Neomycin (neo)

Question 43

Name recessive selectable markers.

Answer 43

hprt and tk.

Question 44

What is the Cre-loxP system?

Answer 44

A site-specific recombination system using Cre recombinase and loxP sites.

Question 45

What is the length of the loxP recognition sequence?

Answer 45

34 base pairs.

Question 46

Does Cre recombination require ATP?

Answer 46

No.

Question 47

What is a chimera?

Answer 47

An animal composed of cells from two or more embryos.

Question 48

How are chimeric mice generated?

Answer 48

Injection of targeted ESC into blastocysts.

Question 49

Purpose of knockout animals?

Answer 49

Study gene function and model human diseases.

Question 50

Name diseases modeled using KO animals.

Answer 50

Cystic fibrosis

Question 51

What are cell-free protein expression systems?

Answer 51

Rabbit reticulocyte lysates

Question 52

Name mammalian expression systems.

Answer 52

Viral vectors and stable cell lines (CHO

Question 53

What is a fusion protein?

Answer 53

A protein expressed fused to a tag for stability and purification.

Question 54

What is a His-tag used for?

Answer 54

Affinity purification using nickel columns.

Question 55

Name common protein tags.

Answer 55

His

Question 56

What is the Sanger sequencing method?

Answer 56

Chain termination sequencing using ddNTPs.

Question 57

What is the function of ddNTPs?

Answer 57

Terminate DNA chain elongation.

Question 58

How are Sanger sequencing products separated?

Answer 58

Gel electrophoresis.

Question 59

How are nucleotides represented in chromatograms?

Answer 59

G=green