DNA Techniques

Question 0

How did the somatic cell nuclear transfer work for Dolly the Sheep?

Answer 0

Nucleus from mammary micro injected into enucleated unfertilized oocyte, zygote transferred to pseudopregnant female, exact genetic copy of the nuclear donor animal developed

Question 1

Define clone and give examples.

Answer 1

Simply an exact genetic copy

Bacteria are clones since they reproduce by binary fission
Dolly the sheep is a clone of her “mother”

Question 2

What is recombinant DNA?

Answer 2

Refers to joining of a DNA sequence of interest to DNA originating from another source (plasmid of bacteriophage)

Question 3

What does recombinant DNA allow?

Answer 3

This allows the isolation and amplification of a specific region of chromosomal DNA (cloning); once amplified, detailed analyses of the DNA structure/protein function are possible

Question 4

What is the basis for most recombinant DNA techniques?

Answer 4

Complementary base pairing

Question 5

What are some sources for DNA for cloning?

Answer 5

Whole genomic DNA
Chromosome specific DNA
cDNA/tissue specific cDNA

Question 6

How is cDNA synthesized?

Answer 6

Using a retro viral enzyme (reverse transcriptase) to make DNA copies of mRNA.

Question 7

What are vectors and what are some examples of vectors?

Answer 7

They are molecules that can replicate autonomously in a host.

Some examples include:
Plasmids
Bacteriophage
Cosmids
Yeast artificial chromosomes (YAC)

Question 8

What does the lyric cycle do to the host?

Answer 8

Kills it

Question 9

What does the lysogenic cycle result in?

Answer 9

Production of dormant prophage

Question 10

What are some bacterial enzymes used in cloning?

Answer 10

Type II restriction endonucleases

DNA ligase

Question 11

What do type II restriction endonucleases do?

Answer 11

They are a bacterial defense mechanism

They recognize 4-8 bp palindromes and cleave them often producing cohesive “sticky” ends

Question 12

What happens when a plasmid is introduced to a new host

Answer 12

Transformation

Question 13

What happens when a bacteriophage is introduced to a host?

Answer 13

Transduction

Question 14

What is transformation?

Answer 14

A natural mechanism of bacterial genetic transfer

Question 15

What is transduction?

Answer 15

Infect bacteria with recombinant virus

Question 16

What happens in phage lambda cloning?

Answer 16

Genes for viral integration into host genome (lysogenic life cycle) are removed and replaced by foreign DNA

Question 17

What do recombinant phases from phage cloning do?

Answer 17

They are infective and will form plaques

Question 18

What are some commonly used probes in probe design?

Answer 18

Cloned DNA fragment from another species
Antibody specific for gene product
PCR amplified DNA fragment
Synthesized DNA fragment based on protein sequence

Question 19

What does sequencing DNA tell you?

Answer 19

Determines protein sequence which can give clues about protein function

Question 20

What do we use expressed recombinant proteins for?

Answer 20

It’s how we get things like insulin, human growth hormone and clotting factors

Question 21

Can you use cloned DNA as a probe?

Answer 21

Yes, it can be used in the detection of mutations in genes (prenatal)

Uses as minisatellite sequences to produce DNA fingerprints (forensics)

Question 22

How can cloned DNA be used in functional studies?

Answer 22

Detect mRNA expression of the gene in tissues, individual cells, in response to signaling molecules

Question 23

How can cloned DNA be used in gene therapy?

Answer 23

Transfer it into the genome to correct a genetic defect

Question 24

What is one method of DNA sequencing?

Answer 24

Sanger Dideoxy (ddNTP) Method

Question 25

How does the Sanger Dideoxy Method work?

Answer 25

DdNTP analogs inhibit DNA polymerase as it synthesizes the complementary strand

Question 26

How was the genome project conducted?

Answer 26

Fully automatically at 1000 bases/sec

Question 27

What is subcloning?

Answer 27

Once isolated, a cloned gene can be excised from the cloning vector and transferred to a different, more specialized vector

Question 28

What can subclones be used for?

Answer 28

Site-directed mutagenesis Fusion protein production In vitro translation Production of recombinant proteins

Question 29

What is site directed mutagenesis used for?

Answer 29

Used to map functional domains of the gene products

Question 30

How do expression vectors work?

Answer 30

Specialized plasmids are used to express proteins in bacteria. This is where we get things like insulin and hGH.

Question 31

What is green fluorescent protein used for?

Answer 31

It is a fusion protein used to tag proteins to monitor when and where they are expressed in an organism

Question 32

What is southern blotting used for and how does it work?

Answer 32

It is used for forensic analysis. Repetitive element probes are used to identify individual patterns

Question 33

What is RFLP analysis used for?

Answer 33

Prenatal diagnosis using the concept of linkage

Question 34

What is microarray technology useful for?

Answer 34

Comparing genes expressed in 2 populations of cells or tissues

Question 35

What is siRNA useful for?

Answer 35

For knocking down protein levels and monitoring effect on cell behavior

Question 36

What is polymerase chain reaction (PCR) useful for?

Answer 36

Making copies for further analysis by allowing selective amplification of a single molecule of DNA several million fold in just a few hours

Question 37

Does the human genome project support use of PCR for gene cloning?

Answer 37

Yup

Question 38

How many DNA regions are tested and stored in CODIS for forensic analysis.

Answer 38

13 matches

Question 39

What are some challenges for proper expression in gene transfer?

Answer 39

Vectors must contain eukaryotic elements Gene must be integrated into host chromosome Site of integration is mostly random

Question 40

What is a knockout?

Answer 40

If introduce inactive gene and homologous lay recombine it into correct site in genome.

Question 41

What is "pharming"?

Answer 41

The term used for introducing genes for human proteins or pharmaceuticals into traditional farm animals and harvesting the products

Question 42

What is the most reliable form of gene therapy and how does it work?

Answer 42

Ex vivo: performed on cells that can be removed from the patient, manipulated and returned.

Question 43

How does in vivo gene therapy work?

Answer 43

Corrected gene is injected into patient via disabled retro viral vectors for the most part

Question 44

What are some non-viral delivery systems for gene delivery?

Answer 44

Liposomes Non-lipid coats Naked DNA Mini chromosomes

Question 45

Is it legal to perform gene therapy on germ cells or embryos?

Answer 45

Nope

Question 46

What optometry related disease has gene therapy been used to treat?

Answer 46

Choroideremia

Question 47

How has gene therapy been used in dentistry?

Answer 47

``` Bone regeneration Periodontal ligament regeneration Dentin regeneration Salivary gland regeneration Oral cancer treatment ```

Question 48

Describe totipotent stem cells.

Answer 48

From zygote | Can form whole human being

Question 49

Describe pluripotent stem cells.

Answer 49

From blastocyst | Can form cell types from all 3 germ layers

Question 50

Describe multipotent stem cells.

Answer 50

From adult | Can form many closely related cell types