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EOM 1 - Test 2 > Brazeau - Recombinant DNA > Flashcards

Brazeau - Recombinant DNA Flashcards

1
Q

What are we looking to identify when we begin understanding gene expression?

A

Looking to isolate mRNA to understand protein

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2
Q

Restriction Endonuclease (RE)

A

Enzymes that cleave DNA at specific sequences

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3
Q

What PTM can block Restriction Endonucleases (RE)s?

A

Methylation

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4
Q

What is the general outline for using Restriction Endonucleases?

A
  1. Cut in specific location leaving overhangs 2. Use ligases to connect 3. Separate via gel electrophoresis
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5
Q

In gel electrophoresis, what is being used to separate?

A

Size, DNA is negatively charged. It will move towards the positive electrode. Gel is porous, so smaller fragments will move farther.

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6
Q

What is a limitation of Restriction Endonucleases?

A

For larger DNA molecules, RE digestion does not provide enough resolution

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7
Q

What enzyme can seal single stranded tails created by REs?

A

DNA ligase

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8
Q

How is recombinant DNA produced?

A
  1. Create fragment w/REs 2. Use DNA ligase to seal fragment in vector DNA
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9
Q

Molecular Cloning

How can fragments be later isolated?

A

DNA fragment inserted into a vector (dna molecule) that can replicate independently in a host cell

Restriction Endonuclease digestion and gel electrophoresis (grind up them little buddies, extract the DNA, chop it up, purify it)

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10
Q

In molecular cloning what do we need to select for our gene?

A

Ampicillin resistant genes

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11
Q

What are two drawbacks for using Vectors for Cloning?

What are some positives?

A
  1. Amount of DNA we can insert is low (1000 bp)
  2. Bacteria have different genes and pathways, so we have to account for differences

(+)

  1. We can move the gene in, and let our bacteria slaves make the protein
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12
Q

What vectors are designated to accomodate large DNA inserts?

A

Cosmids (bacteriophage)

BACs

YACs

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13
Q

What enzyme is used to clone RNA?

What is the result?

What does this allow us to explore?

A

Reverse Transcriptase

cDNA

noncoding sequences in eukaryotic genes

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14
Q

What is a mathematical result of the relative low cost and speed we can sequence DNA?

A

Massive data generation, need to be able to process/analyze it

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15
Q

Whole Exome Sequencing

A

All of the (coding) exonds in the genome, allows us to better study introns and finding differences in humans

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16
Q

What do we look to exclude when RNA Sequencing?

A

Get rid of housekeeping genes

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17
Q

What do Chip Seq-DNA let us figure out?

A

GIve data on what DNA sequence proteins bind to

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18
Q

Genomic Medicine

A

Monitor and guide therapy, compare genome of patient to genome of tumor

19
Q

How do we use sequencing in epigenetics?

A

Use DNA to isolate where methylated, looks for non methylated cytosines and compare two sequences.

For example, that of a crack-baby to a normal baby

20
Q

Metagenomics

Example?

A

Studying the sequence of the microbiome

Dutch famine study

21
Q

What gene is vital for metagenomics?

What is special about it?

What can it tell us?

A

16s rRNA gene

Highly conserved in all bacteria, but also has regions of variable sequences; allows to ID bacteria at the species level

22
Q

What is a technology based on high throughput sequencing that can give a sequence without culturing?

A

Metagenomics

23
Q

What are the steps for Nucleic Acid Hybridization?

A
  1. Denature
  2. Add labeled DNA probe complementary to specific sequence
  3. Renature
24
Q

Southern Blot

A

Used to identify DNA expression

25
Q

What is photolithography (Affymetrix) still used for?

A

Genotyping

26
Q

What is the general process for PCR?

A

Take a pile of DNA, amplify specific sequence, clone it

  1. Denature
  2. Anneal
  3. Extension
  4. Repeat
27
Q

What does PCR require to run?

A
  1. 2x flanking oligonucleotide primers
  2. Thermo-stable DNA polymerase
  3. dNTP’s
  4. Target DNA
  5. Repeated cycles
28
Q

What is one method of assessing gene expression?

A

Real-time PCR (Quantitative PCR)

Formation of product is continuously monitored

29
Q

in situ PCR

A

Running PCR on a slide; can yield what tissue a gene is expressed in

30
Q

Gene Therapy

Draw back?

A

Transfer of genes into other organism to turn proteins on/off

( - ) Foreign DNA is not introduced into genome–only get short-term expression

31
Q

Transgenic Mice

A

Mice that carry foreign genes, produced by microinjection of cloned DNA into pronucleus of fertilized egg

32
Q

What methods are used to get cloned genes into mice?

Which is easier?

A
  1. Microinjection into nucleus
  2. Embryonic Stem Cells (ES)

ES is easier. Cloned DNA introduced into ES cells in culture, then transformed cells are introduced back into mouse embryos

33
Q

What is a result of Embryonic Stem (ES) Cell mice?

What can breeding yield?

A

Produce Chimeric mice

Breeding can yield homozygotes

34
Q

To determine role of cloned gene, what can we do to a normal gene copy?

A

Knock it out

“Gene Knockout”

35
Q

What normal function of DNA replication are you taking advanatges of in gene knockout?

A

Homologous Recombination

36
Q

Cre-Lox System

What does this allow for?

A

Takes advantage of site-specific recombinase to introduce gene deletions, inversions, or translocations

Conditional Mutagenesis

37
Q

Conditional Mutagenesis

A

Ability to delay expression to a later time, or a specific place

38
Q

What does CRISPR/Cas System allow?

A

Allows Regulation Expression

39
Q

For CRISPR edits to be implemented at the organism level, what would have to occur?

A

Would have to make the edits early, at the embryo level

40
Q

What does RNA Interference give us the ability to do?

A

Turn off mRNA, thus proteins

41
Q

What type of RNA allows posttranscriptional inhibition (Transcriptional Gene Silencing (TGS))?

A

Small Interfering RNA

42
Q

What is the manipulation of an inherent system to block RNA transcription?

What can these do?

Two drawbacks?

A

siRNA

Turn off genes

  1. How do we target specific spots?
  2. We have a lot of RNAse, which degrades these
43
Q

siRNA

What level do they work at and how?

A

Small Interfering RNA

Post-transcriptionally, in a catalytic manner and very low concentrations

44
Q
A

EOM 1 - Test 2 (18 decks)

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