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Recombinant DNA/biotechnology Flashcards

1
Q

What are the requirements for cell based DNA cloning (specific amplification)?

A
  1. Suitable host cell
  2. Replicón
  3. Means of attaching foreign DNA to replicon and transferring to host cell
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2
Q

What are the requirements for polymerase mediated in vitro DNA cloning (PCR)? (Specific amplification)

A
  1. Thermostable DNA polymerase

2. Sequence information enabling synthesis of specific oligonucleotide primers

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3
Q

What are the requirements for molecular hybridization (specific detection)

A
  1. Labeled DNA or RNA probe

2. Means of detecting fragments to which probe binds

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4
Q

What kind of endonucleases are restriction enzymes?

A

Type II endonucleases

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5
Q

______ enzymes cut at specific sequences of 4- to 8- base pairs called ______ to give staggered or blunt-ended DNA fragments.

A

Restriction, “restriction site”

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6
Q

How are restriction enzymes named?

A

According to bacterium they are isolated from and then numbered

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7
Q

What does the frequency of a restriction site in a given DNA sequence depend on?

A

The length of recognition

I.e.
– 4 bp restriction site: fragments average 256 bp
– 6 bp restriction site: fragments average 4096 bp
– 8 bp restriction site: fragments average 65,536 bp

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8
Q

Where do restriction endonucleases cut?

A

In palindromes

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9
Q

What are restrictions enzymes used for?

A
  1. Identify DNA fragments and mutations

2. Create new gene constructs

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10
Q

Any mutation in restriction site makes it _____ or a new site appears.

A

Uncuttable

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11
Q

How are new gene construct made by restriction enzymes?

A

cutting out
specific DNA fragments – that are then ligated
back into a cut vector DNA to:

a) purify the fragment (bycloning),
b) sequence the fragment, or
c) express them in vitro (transfection into cells) or in vivo (make transgenic animals, plants, yeast, – etc).

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12
Q

Analysis of DNA by ___ gel is usually blotted for analysis

A

“Restriction mapping”

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13
Q

What does a restriction map represent?

A

A linear sequence of the restriction enzyme sites on the target DNA

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14
Q

Distances along restriction maps are measured directly in?

A

Base pairs (bp), kilobase-pairs (Kbp), or mega base pairs (1Mbp= 1 million bp)

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15
Q

What is Southern blotting used for?

A

DNA

Note: SNoW DRoP
S= DNA
N= RNA
W= Protein

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16
Q

what detect DNA sequence within a mixture of fragments?

A

Specific probes

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17
Q

___ depends on the re-annealing properties of DNA

A

Molecular hybridization

I.e.
Hybridization in 50% for amide at 42*C only forms 1 stable double helix

Hybridization in 50% formamide at 35*C forms 3 stable double hélices

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18
Q

The KARP-1 LR DNA is ______ specific

A

Primate

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19
Q

The ___ DNA is strongly conserved in evolution

A

Ku86

Note: restriction fragment size differ between species

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20
Q

Zoo blot analysis DNA hybridization is used to?

A

Investigate interspecies conservation

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21
Q

What is the process of DNA fingerprinting with southern blots?

A
  1. Cleave Chromosomal DNA with restriction endonucleases
  2. Separate DNA fragments by agarose gel electrophoresis (unlabeled)
  3. Denature DNA, and transfer to nylon membrane
  4. Incubate with probe, then wash
  5. Expose X-ray film to membrane
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22
Q

What restriction site does sickle cell mutation change?

A

MstII

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23
Q

What doe the restriction endonuclease MstII recognize and cleave?

A

The sequence CCTNAGG (where N is any nucleotide)

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24
Q

What codon is changed in sickle cell?

A

The codon for the 6th amino acid in B-globin gene

Note: the A to T mutation within Odin six of the B^s-globin gene eliminates a cleavage site for the enzyme, MstII

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25
Q

Electrophoresis of restriction fragments from the DNA of a normal individual yields a ____ fragment using a probe specific for the B-globin gene.

A

1150bp

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26
Q

Electrophoresis of restriction fragments from the DNA of a sickle cell individual yields a ____ fragment using a probe specific for the B-globin gene.

A

1350 bp

Note: this is because of the loss of a cleavage site

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27
Q

Electrophoresis of restriction fragments from the DNA of a heterozygote for sickle cell yields ____ fragments.

A

Both 1150bp and 1350 bp

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28
Q

A southern blot of BamHI/DNase digested DNA use what radio labeled probe?

A

Globin cDNA

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29
Q

What does Northern Blotting analyze?

A

RNA

Note: SNoW DRoP

S= DNA
N= RNA
W= Protein
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30
Q

What process is this describing?

  1. RNA was isolated from nuclei and cytoplasm separately.
  2. The RNA was separated according to size, then blotted onto membrane.
  3. This blot was hybridized with a labeled cDNA probe specific to a particular sequence in the mRNA of interest
  4. This results show the process of RNA-splicing
A

Northern blotting

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31
Q

___ based systems have nearly replaced Northern and southern blots.

A

Microarray

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32
Q

______ blots: fluid phase single probe and immobilized complex target.

A

Southern and northern

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33
Q

What allows for multiple immobilized target and complex probes (

A

Microarray

Note: the array system allows for an unlimited number of parallel assay but you need different labels

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34
Q

What is replacing northern blots?

A

Expression arrays

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35
Q

Why would you expect to see larger sized RNAs in the nuclear store the bind to your specific probe?

A

These larger RNA’s represent the splicing intermediates that arise during maturation of mRNA

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36
Q

What has replaced southern blotting in almost all applications?

A

Genomic arrays

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37
Q

_____ has become extremely fast and good; conventional DNA sequencing is still used for economic reasons

A

Array-based sequencing

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38
Q

What is a collection of microscopic DNA spots attached to a solid surface?

A

DNA microarray (aka, Gene chip, DNA chip, Bio-chip)

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39
Q
  1. Isolate mRNAs from cells at two stages of development; each mRNA sample represent all the genes expressed in the cells at that stage
  2. Convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxyribonucleotide triphosphate
  3. Add the cDNAs to a microarray; fluorescent cDNAs anneal to complementary sequences on the microarray
    4 each fluorescent spot represents a gene expressed in the cells

What process was just done?

A

Expression array

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40
Q

In expression array, each mRNA sample represents ?

A

All the genes expressed in the cells at that stage

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41
Q

In expression arrays, what converts mRNs to cDNAs? (Second step)

A

Reverse transcriptase

Note: they use labeled deoxyribonucleotide triphosphates for this

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42
Q

What does mamma print microarray measure?

A

The activity of 70 genes to assess risk of breast cancer metastasizing

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43
Q

In mammaprint, what are the classification of threshold ?

A

High risk or low risk, no intermediate

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44
Q

___ enzymes are endonucleases for double stranded DNA and are strictly sequence-specific. The fragments produced are called restriction fragments

A

Restriction enzymes

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45
Q

Because restriction enzymes can digest DNA from any source, DNA from ___ can be pieced together

A

Different species

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46
Q

A given DNA fragment can be characterized by the location of the restriction sites in the?

A

Restriction map

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47
Q

What joins DNA fragments treated with the same restriction enzyme?

A

DNA ligase

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48
Q

____ blotting is the technique to identify and size restriction fragments in a complex DNA mixture by DNA-DNA hybridization

A

Southern

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49
Q

In the _____ the separated DNA fragments are denatured and
transferred to an easily managed membrane allowing a very sensitive
detection with a labeled probe. (Southern blotting)

A

Blotting step

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50
Q

The ______. used in the hybridisation step can be anywhere
between 20 and 2000 bases long and the longer the probe the more
mismatches can be allowed between probe and target (southern blotting)

A

single stranded probe

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51
Q

For probes based on _____ sequence, a region in the polypeptide is identified with the least possible codon usage. (Southern blotting)

A

Amino acid

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52
Q

_______ blotting is a method to identify and size mRNA’s in a complex
mixture by DNA/RNA hybridisation.

A

Northern

Note: The procedure is similar to Southern
blotting but restriction enzymes are not used

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53
Q

______ are small glass plates with multiple

spots containing a defined DNA sequence

A

DNA micro Arrays (aka gene chips)

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54
Q

In __________ the arrayed DNA sequences are 50 nucleotides long
and are identical to selected mRNA sequences (except T’s for U’s) and in
genomic arrays the arrayed sequences are identical to selected
chromosomal DNA sequences (as equally spaced as possible)

A

Expression arrays

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55
Q

What can be compared in expression arrays by
incorporating different fluorescently labeled deoxynucleotides in the reverse
transcription step ?

A

Different complex mRNA mixtures

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56
Q

Expression profiling is becoming an important tool to characterize ?

A

diseased tissue such as staging of tumors

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57
Q

How is comparative genomic hybridization done? (CGH or ultra sensitive cytogenetic)

A
  1. Cell from patient and cell from normal control
  2. Extract DNA
  3. Label with two different fluorochromes
  4. Mix in equal quantities; hybridized to microarray of clones
  5. Read red and green fluororescene (test DNA green and control DNA red)
  6. Work out red:green ratio of each cell; align to database of clones
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58
Q

What is being described?
1. Take restriction-cut DNA fragments

  1. Ligate them into a bacterial vector (a modified bacterial plasmid)
  2. Transfect recombinant plasmid into a bacteria
  3. Select for correct antibiotic resistance in growth medium
  4. Plate out – and – each colony is a clone of one original bacterium containing one DNA fragment
A

It’s a summary of the original version of DNA cloning

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59
Q

Staggered and bound ends can be rejoins (“lighted” - combining different DNA fragments = ?

A

Recombinant DNA

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60
Q

How is recombinant DNA cloning done?

A
  1. Cloning vector is cleaved with restriction endonuclease
  2. DNA fragment of interest is obtained by cleaving chromosome with a restriction endonuclease
  3. Cut fragments and vector are mixed and allowed to anneal
  4. Fragments are lighted to the prepared cloning vector
  5. DNA is introduced into the host cell
  6. Transformed bacteria are plated onto a selective medium
  7. Propagation (cloning) produces many copies of recombinant DNA
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61
Q

In DNA cloning what combines the DNA fragments and vectors together?

A

DNA ligase

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62
Q

How can you use pBR322 to clone foreign DNA? (Step 1-3)

A
  1. pBR322 is cleaved at the ampicillin-resistance element by Pstl.
  2. Foreign DNA is ligares to cleave pBR322. Where ligation is successful, the ampicillin-resistance element is disrupted. The tetracycline-resistance elements remain intact.
  3. E. coli are transformed, then grown on agar plates containing tetracycline to select for those that have taken up plasmid
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63
Q

How can you use pBR322 to clone foreign DNA? (Step 4-5)

A
  1. Individual colonies are transformed to matching positions on additional plates. On plate contains tetracylcine, the other tracycline and ampicillin
  2. Cells that grow on tetracycline but not on tetracycline + ampicillin contain recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA. Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates
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64
Q

What cleaves pBR322 at the ampicillin resistant element Pstl?

A

Pstl restriction endonuclease

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65
Q

What are the roles of antibiotics in cloning?

A
  1. Eliminate bacteria that have not taken up a plasmid
  2. Identify bacteria that have taken up a plasmid without an insert (with second antibiotic)
  3. Increase the number of plasmids in the bacterium from 3-4 in the dormant state to thousands when challenged by the antibiotic
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66
Q

______ identify bacterial colonies that contain the insert of interest. (When finding a particular DNA fragment)

A

Positive spots

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67
Q

___ with a specific piece of labeled DNA (made from part of the known gene or predicted from aa sequence)

A

Probe library (for DNA fragments)

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68
Q

_____ probe is hybridized against a filter
onto which the DNA of many different colonies has
been spotted & fixed

A

Specific labeled

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69
Q

How do you use hybridization to identify a clone with a particular DNA segment?

A

(Start with an agar plate with transformed bacterial colonies)
1. Press nitrocellulose paper onto the agar plate. Some cells from each colony stick to the paper.

  1. treat with alkali to disrupt cells and expose denatured DNA
  2. Insert radiolabeled DNA probe and incubate the paper with the radiolalbeled probe, then wash.
  3. Expose to x-ray film
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70
Q

How do you prepare a cDNA library?

A
  1. Lose cells and purify mRNA
  2. Hybridize with poly (T) primer
  3. Make DNA copy with reverse transcriptase
  4. Degrade RNA with RNase H
  5. Synthesize a complementary DNA strand using DNA polymerase; RNA fragment acts as a primer
    Product: double-stranded cDNA copy of original mRNA
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71
Q

What is the difference between the genomic and cDNA library?

A

Genomic DNA clones the entire DNA

CDNA clones are clones of the mRNA (post transcription)

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72
Q

What enzymes are used to make a genomic library?

A

Restriction endonuclease

DNA ligase

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73
Q

What enzymes are used to make the cDNA (expression) library?

A

Reverse transcription

DNA ligase

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74
Q

What kind of library constrain nonexpressed sequences of chromosomes?

A

Only genomic libraries

75
Q

What kind of genomic libraries contain introns, promoter and enhancer sequences?

A

Only genomic libraries

Note: promoter and enhancer may not be in the same clone

76
Q

Genes in what library can be expressed in cloning host? (Recombinant proteins)

A

cDNA (expression) libraries

77
Q

What load of libraries can be used for gene therapy or for constructing transgenic animals?

A

cDNA (Expresión) libraries

Genomic libraries are yes and no

78
Q

What kind of vectors are used in the cDNA library?

A

Plasmid (< 15kbp)

Bacteriophage (<23 Kbp)

79
Q

What kind of vectors are used in genomic libraries?

A

Bacterial artificial chromosome (<300 kbp)

Yeast artificial chromosome (<2000 kbp)

80
Q

How is subcloning of a DNA sequence for expression done?

A 
Start with double-stranded plasmid DNA expression vector
1. Cut DNA with restriction nuclear
2. Insert protein-coding DNA sequence
3. Introduce recombinant DNA into cells 
Results: over expressed mRNA and protein
81
Q

Insulin in humans is derived from a precursor

polypeptide called?

A

Proinsulin

82
Q

______ folds itself by specific disulfide

bridges followed by proteolytic processing

A

Proinsulin

83
Q

What is unable to process human proinsulin these can produce both chains?

A

Bacteria

Note: Upon mixing of the two chains and subsequent
oxidation to form the disulfide bonds active human insulin is generated

84
Q

By how many does PCR increase each increment by?

A

It doubles each increment

(2^n), n= cycle no.,

85
Q

What cycle does PCR go through?

A
  1. Denaturing
  2. Annealing
  3. Elongation

Repeat
Repeat

86
Q

Making recombinant proteins in eukaryotic cells _____ cDNA encoding protein of choice from cDNA library

(I.e., cut the cDNA from vector w/ restriction enzymes)

87
Q

Making recombinant proteins in eukaryotic cells _____ cDNA into polylinker of expression vector cut with compatible restriction enzyme

88
Q

Selecting for Neo^R (G418 in media). Neo^R is a prokaryotic gene so must add a promoter and poly A site (SV40 enhancer & pA). cDNA is expressed from CMV promoter. What does this do?

A

Transforms eukaryotic cells

89
Q

What does synthetic Neo do to eukaryotic cells?

A

Kills them, it’s why Neo^R is selected (G418 in media )

90
Q

Where does the gene for the enhancer region and poly A tail come for eukaryotic recombinant proteins

A

SV40 enhancer and pA

91
Q

Where does the promoter come from when making recombinant proteins in eukaryotic cells ?

A

cDNA is expressed from CMV promoter (polyA from the BGH gene )

92
Q

What PCR product is used for haemophila A and B?

A

Factor VIII, IX

93
Q

What PCR product is used for Hairy-cell leukaemia?

A

Alpha- interferon

94
Q

What PCR product is used for multiple sclerosis?

A

Beta-interferon

95
Q

What PCR product is used for infections in CGD?

A

Y-interferon

96
Q

What PCR product is used for Gaucher disease?

A

Glucocerebrosidase

97
Q

What PCR product is used for thrombosis?

A

Tissue plasminogen activator (TPA)

98
Q

What PCR product is used for obesity?

A

Leptin (has not worked out)

99
Q

What PCR product is used for anemia?

A

Erythropoietin

100
Q

What PCR product is used for Type II Diabetes?

101
Q

What do Poly-His leaders bind reversibly?

A

Nickel ion

102
Q

What’s the process of making recombinant proteins in eukaryotic cells?

A
  1. Purify
  2. Lígate
  3. Transform E. coli
  4. Transform eukaryotic cells
103
Q

When transforming E. coli in making recombinant proteins in eukaryotic cells, what is selected for (why?)?

A

Select for AP^R to obtain sufficient plasmid DNA and check sequence, orientation of cDNA

104
Q

What does GFP do to tissue?

A

Green fluorescent protein (GFP) makes things glow. . .

105
Q

What s used to determine the promotor elements responsible for the pattern of gene’s expression?

A

Reporter protein

106
Q

________ uses chromosomal clone

arrays to detect deletions or duplications & altered expression

A

Comparative genome hybridization

107
Q

Bacterial plasmids are extrachromosomal circular elements carrying ________; selecting with an antibiotic will increase plasmid number from 3-4 to possibly1000copies per cell !

A

antibiotic resistance genes

108
Q

_____ can be used to select for bacteria which have a plasmid with an inserted DNA fragment

A

Antibiotic resistance

109
Q

By plating out bacteria harboring a plasmid with an DNA insert you can get a ___ that is propagated to prepare lots of plasmid DNA.

A

pure clone colony

110
Q

_____ are characterized by restriction mapping or DNA sequencing

111
Q

cDNA libraries are made from ____ which has been converted to cDNA with the help of reverse transcriptase and RNaseH.

112
Q

The primer for the reverse transcriptase is ____that works as an universal primer as all mRNA’s have a polyA tail

A

oligo dT (a stretch of about 20 T’s)

113
Q

_____ clones contain DNA corresponding only to exons and _____ clones can contain any type of genomic DNA such as introns, exons and/of intergenic regions.

A

cDNA

Genomic DNA

114
Q

Human insulin is commercially produced in bacteria by making
the A and B peptides separately normally and then joining by
____

A

oxidizing the cysteine side chains

115
Q

Using a reporter gene, you can?

A

Determine the promotor elements responsible for the pattern of a genes expression

116
Q

Animals that are genetically engineered by gene insertion, deletion or replacement are?

A

Transgenic organisms

117
Q

What are transgenes?

A

Any foreign of modified genes added

118
Q

When is an animal considered a knock out?

A

When both copies of the gene are completely inactive or deleted

119
Q

If a mutated gene is transferred into a cell it usually integrate at ____ into the cell.

A

Random

Note: one in thousand times it replaces one of the
copies of the normal gene by “homologous recombination”

120
Q

What are gene replacements used for?

A

To study the effect of point mutations

Note: only active gene is active

121
Q

What are gene knockout used for?

A

To study the effect of gene deletions

Note: no active gene is present

122
Q

What are gene addition used for?

A

To study the dominant negative mutation or inducible gene systems

Note: both normal and mutant genes are active

123
Q

What animals are used to study gene expression during development?

A

Mice, zebrafish, flies and worms

124
Q

What is farmogenics?

A

Producing pharmaceuticals in transgenic animal (sheep, goats, cattle, pigs and rabbits)

125
Q

What is an example of a transgenic animal?

A

Milk a cow – and the milk has Tissue Plasminogen Activator in it

126
Q

How are genes added by micro injection?

A
  1. Have a fertilized egg
  2. Wash fertilized eggs out of oviducts
  3. Microinject gene of interest (foreign DNA) into pronucleus
  4. Implant eggs into psuedopregnant mouse
127
Q

Strong conservation of what throughout evolution suggests role in gene regulation?

A

Non-coding sequences (multi-species )

Ex: LMX1B

128
Q

Each construct is injected into fertilized eggs to generate lines of transgenic mice (or different cell lines) -then compare levels of reporter gene expression in different lines to localize promoter/enhancer elements required for expression in different tissues

What process is being done?

A

Mapping promotor elements in transgenic animal

129
Q

What is the myc oncogene under control of?

A

Tetracycline

130
Q

What two artificial genes are used in inducible gene expression?

A

Myc oncogene and gene for a hybrid transcription factor

131
Q

Tetracyclin acts here as a repressor for ______ because upon binding to the hybrid transcription factor it causes the factor to dissociate from the promotor

A

Myc oncogene

132
Q

What are these used for?
The myc oncogene under control of a Tetracycline
responsive (promotor) element TRE (tetracycline is used here as a signal molecule, not as an antibiotic)

Gene for a hybrid transcription factor consisting of a domain
which binds to the TRE and a domain that directly activates the RNA polymerase under control of a promotor which only works in white blood cells

A

Inducible gene expression

133
Q

What is the essential tool for gene knock out and gene replacement?

A

Embryonic stem (ES) cells

134
Q

Inbreeding of the mouse each carrying one mutant copy of the gene produces homozygous mice where both copies are mutated. In case the mutant genes do not produce a functional protein the homozygous mice are called

A

“knock-out” mice

135
Q

Describe gene modification in mice.

A
  1. DNA into ES cells in culture and then transformed
  2. Inject into blastocyst
  3. Inject embryos to foster mothers. These mice are chimeric
  4. Breed chimeric mice to produce transferred gene offspring (earn gene is in germ line)
136
Q

What is gene targeting?

A

Positive and negative selecto to select for homologous recombinant in transgenic animals

137
Q

What is a positive selection for gene-targeting?

A

An exon from a targeted gene is disrupted by the a
neo gene containing its own promotor encoding
resistance to the toxic agent G418

138
Q

An exon from a targeted gene is disrupted by the a
___ gene containing its own promotor encoding
resistance to the toxic agent G418

A

Neo

Positive selection

139
Q

What is downstream from the neo gene?

A

A tk (thymidine Kinase) gene

140
Q

Downstream of the neo gene is a tk (Thymidine
Kinase) gene, that when integrated and expressed
in the host genome provides the target for the toxic
agent gancylovir

What process is being described?

A

Negative selection

141
Q

Downstream of the neo gene is a tk (Thymidine
Kinase) gene, that when integrated and expressed
in the host genome provides the target for the toxic
agent ________

A

Gangylovir

Note: Neo+ tk- colonies frequently carry the desired mutation

142
Q

What kind of colonies frequently carry the desired mutation?

143
Q

What does the absence of Lmx1b affect the development of?

A

Dorsal limb structures

144
Q

When you have a nonhomologous recombination (for gene targeting), the cell is?

A

G418 resistant and gancylovir sensitive

145
Q

When you a homologous recombination (for gene-targeting events), the cell is?

A

(Target gene is disrupted) and cell is g418 resistant and gancylovir resistnat

146
Q

Insertion of what inactivates the gene lmx1b?

A

Targeting PGK-Neo

147
Q

____ allow for a rapid and specific method to amplify sequences of interest
nearly a billion-fold

148
Q

PCR-amplified sequences can be used for ____

A

almost anything

149
Q

In vectors used for expression in eukaryotic cells a eukaryotic promoter and a polyadenylation signal are required, and the _____ must work in eukaryotic cells

A

resistance gene

150
Q

_____ can be expressed in eukaryotic cells, isolated, and tested for function

A

His-tagged proteins

151
Q

In specialized cDNA libraries the cDNA insert is fused to another reporter gene to study ______

A

protein function

152
Q

______ can be fused with the gene fragment that codes for GFP protein for whole-animal or intracellular protein localization studies

A

cDNA or genomic DNA

153
Q

______ are animals in which a gene is replaced, knocked out or in which a gene is added

A

Transgenic animals

154
Q

Genes can be added by _____ of the gene constructs in the pronucleus of fertilized eggs

A

microinjection

155
Q

To test a chromosomal DNA fragment for ____ it is fused to a reporter gene and the expression is tested in multiple tissues or in the
experimental animal

A

promotor activity

156
Q

The ___ permits tissue-specific inducible-gene expression.

Tetracycline acts like a switch to prevent undesired expression. Thus, you can study effects of gene-products

A

Tet-off system

157
Q

____is a special recombination in which the target gene exchanges DNA with the introduced gene construct.

A

Homologous recombination

158
Q

By adding the ___ at the end of a gene construct, homologous recombination can be selected for.

159
Q 
The progeny of modified embryo’s are \_\_\_\_\_\_ and further inbreeding is required to select those animals in which also the germ line has
been replaced (by the descendants of the modified stem cells)
A

genetic mosaic

160
Q

How is site-directed mutagenesis carried out?

A

With a cloning vector (w/ inserted normal gene)

  1. Separate strands, use synthetic oligonucleotide primer containing the desired mutated gene
  2. strand completion by DNA polymerase and DNA ligase
  3. Introduction into cells followed by replication and segregation into daughter cells

Two products: normal protein and protein with the single desired amino acid change are made

161
Q

What do transgenic mice with mutant DNA helicase, Xpd(B), show?

A

Premature aging

Note: the point mutation is R722w

162
Q

What point mutation was used to express a mutant XPD gene in mice?

163
Q

What are the strategies for gene therapy?

A
  1. Gene augmentation therapy
  2. Targeted gene mutation correction
  3. Targeted inhibition of gene expression
  4. Direct killing of disease cells
164
Q

What protein is used for gene-editing therapy?

A

CRISPR/Cas-9

165
Q

Buy the Cas9 endonuclease and CRISP guide RNA kit – then ____ to find your target

A

modify the guide RNA

166
Q

When viruses are used for gene therapy, what s done to its genome?

A

Part of the viral genome is deleted to prevent replication

167
Q

_____ vectors do not integrate into the genome and carry larger inserts, but have to be given repeatedly at large doses – can result in immunological problems

A

Adenovirus

168
Q

Successful gene therapy has been achieved in X-;inked adrenoleuko-dystrophy (accumulation of long fatty acids) with?

A

Lentivirus vector (an HIV derivative)

169
Q

How is gene therapy for severe combine immunodeciency carried out?

A
  1. Remove bone marrow
  2. Enrich for progenitor cells by CD-34 antibody conjugated to magnetic beads
  3. Each day for 3 days add gene therapy vector (Maloney mouse leukemia virus containing human IL2RG gene)
    * (some cells transduced)
  4. Infuse cells into patients
170
Q

What’s that deficiency in SCID?

A

ADA (adenine deaminated deficiency)

171
Q

What are interning RNA’s? (iRNA)

A

Sequence-specific mRNA silencing in response to adding in short double-stranded RNA’s

172
Q

What mediates gene silencing which produces a small RNA of 20-30 nucleotides in length that is very similar to the siRNA (for iRNA)

A

Dicer ribonuclease

173
Q

___ can silence endogenous genes or parasitic gens or mutated genes.

174
Q

__ can silence pathogenic invaders such as transposons and viruses

175
Q

What is the main catalytic component of the RISC complex for iRNA and an endoRNase?

176
Q

______ are obtained from a very early immature embryo

A

Embryonic stem cells

177
Q

ICM cells stop dividing after removal from _____ and no longer express stem cell markers. A few days later, some cells may resume growing and re-express stem cell markers - these are ES cells and have no exact embryonic counterpart

A

blastocyst

178
Q 
\_\_\_\_\_ is used for introduction of a point
mutation in a particular gene – can be used to study protein
function (e.g., enzymes)
A

Site-directed mutagenesis

179
Q

_____ is still being perfected; strategies include gene

additions/replacements

A

Gene Therapy

180
Q

DNA can be clinically administered packaged within liposomes

or disabled viruses – and it can be administered by ?

A

electro-poration

181
Q

______ stem cells are obtained from fully differentiated cells
by adding in 3-4 specific transcription factors resulting in complete genetic
reprogramming.

A

Induced pluripotent

182
Q

True pluripotency has to be confirmed by demonstration that the ___ can contribute to any tissue

183
Q

______ from a given patient can be used
to replace the diseased tissue of their own cells or used for disease
modelling to test new therapeutics

A

Induced pluripotent stem cells (IPSC’s)

184
Q

Within the _____ cuts the targeted mRNA minimizing its
translation. Corresponding protein levels decline by 50-60%. In addition, this is now a routine laboratory method using commercial kits.

A

RISC Argonaute

Mcb1 (13 decks)

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