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MCBM Exam 5 Kat > DNA Techniques > Flashcards

DNA Techniques Flashcards

0
Q

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

A

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

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1
Q

Define clone and give examples.

A

Simply an exact genetic copy

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

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2
Q

What is recombinant DNA?

A

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

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3
Q

What does recombinant DNA allow?

A

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

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4
Q

What is the basis for most recombinant DNA techniques?

A

Complementary base pairing

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5
Q

What are some sources for DNA for cloning?

A

Whole genomic DNA
Chromosome specific DNA
cDNA/tissue specific cDNA

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6
Q

How is cDNA synthesized?

A

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

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7
Q

What are vectors and what are some examples of vectors?

A

They are molecules that can replicate autonomously in a host.

Some examples include:
Plasmids
Bacteriophage
Cosmids
Yeast artificial chromosomes (YAC)
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8
Q

What does the lyric cycle do to the host?

A

Kills it

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9
Q

What does the lysogenic cycle result in?

A

Production of dormant prophage

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10
Q

What are some bacterial enzymes used in cloning?

A

Type II restriction endonucleases

DNA ligase

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11
Q

What do type II restriction endonucleases do?

A

They are a bacterial defense mechanism

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

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12
Q

What happens when a plasmid is introduced to a new host

A

Transformation

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13
Q

What happens when a bacteriophage is introduced to a host?

A

Transduction

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14
Q

What is transformation?

A

A natural mechanism of bacterial genetic transfer

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15
Q

What is transduction?

A

Infect bacteria with recombinant virus

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16
Q

What happens in phage lambda cloning?

A

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

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17
Q

What do recombinant phases from phage cloning do?

A

They are infective and will form plaques

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18
Q

What are some commonly used probes in probe design?

A

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

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19
Q

What does sequencing DNA tell you?

A

Determines protein sequence which can give clues about protein function

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20
Q

What do we use expressed recombinant proteins for?

A

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

21
Q

Can you use cloned DNA as a probe?

A

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

Uses as minisatellite sequences to produce DNA fingerprints (forensics)

22
Q

How can cloned DNA be used in functional studies?

A

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

23
Q

How can cloned DNA be used in gene therapy?

A

Transfer it into the genome to correct a genetic defect

24
Q

What is one method of DNA sequencing?

A

Sanger Dideoxy (ddNTP) Method

25
Q

How does the Sanger Dideoxy Method work?

A

DdNTP analogs inhibit DNA polymerase as it synthesizes the complementary strand

26
Q

How was the genome project conducted?

A

Fully automatically at 1000 bases/sec

27
Q

What is subcloning?

A

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

28
Q

What can subclones be used for?

A

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

29
Q

What is site directed mutagenesis used for?

A

Used to map functional domains of the gene products

30
Q

How do expression vectors work?

A

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

31
Q

What is green fluorescent protein used for?

A

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

32
Q

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

A

It is used for forensic analysis.

Repetitive element probes are used to identify individual patterns

33
Q

What is RFLP analysis used for?

A

Prenatal diagnosis using the concept of linkage

34
Q

What is microarray technology useful for?

A

Comparing genes expressed in 2 populations of cells or tissues

35
Q

What is siRNA useful for?

A

For knocking down protein levels and monitoring effect on cell behavior

36
Q

What is polymerase chain reaction (PCR) useful for?

A

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

37
Q

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

A

Yup

38
Q

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

A

13 matches

39
Q

What are some challenges for proper expression in gene transfer?

A

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

40
Q

What is a knockout?

A

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

41
Q

What is “pharming”?

A

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

42
Q

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

A

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

43
Q

How does in vivo gene therapy work?

A

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

44
Q

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

A

Liposomes
Non-lipid coats
Naked DNA
Mini chromosomes

45
Q

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

A

Nope

46
Q

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

A

Choroideremia

47
Q

How has gene therapy been used in dentistry?

A 
Bone regeneration
Periodontal ligament regeneration
Dentin regeneration
Salivary gland regeneration
Oral cancer treatment
48
Q

Describe totipotent stem cells.

A

From zygote

Can form whole human being

49
Q

Describe pluripotent stem cells.

A

From blastocyst

Can form cell types from all 3 germ layers

50
Q

Describe multipotent stem cells.

A

From adult

Can form many closely related cell types

MCBM Exam 5 Kat (15 decks)

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