Brazeau - Recombinant DNA

Question 1

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

Answer 1

Looking to isolate mRNA to understand protein

Question 2

Restriction Endonuclease (RE)

Answer 2

Enzymes that cleave DNA at specific sequences

Question 3

What PTM can block Restriction Endonucleases (RE)s?

Answer 3

Methylation

Question 4

What is the general outline for using Restriction Endonucleases?

Answer 4

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

Question 5

In gel electrophoresis, what is being used to separate?

Answer 5

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

Question 6

What is a limitation of Restriction Endonucleases?

Answer 6

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

Question 7

What enzyme can seal single stranded tails created by REs?

Answer 7

DNA ligase

Question 8

How is recombinant DNA produced?

Answer 8

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

Question 9

Molecular Cloning

How can fragments be later isolated?

Answer 9

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)

Question 10

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

Answer 10

Ampicillin resistant genes

Question 11

What are two drawbacks for using Vectors for Cloning?

What are some positives?

Answer 11

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

Question 12

What vectors are designated to accomodate large DNA inserts?

Answer 12

Cosmids (bacteriophage)

BACs

YACs

Question 13

What enzyme is used to clone RNA?

What is the result?

What does this allow us to explore?

Answer 13

Reverse Transcriptase

cDNA

noncoding sequences in eukaryotic genes

Question 14

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

Answer 14

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

Question 15

Whole Exome Sequencing

Answer 15

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

Question 16

What do we look to exclude when RNA Sequencing?

Answer 16

Get rid of housekeeping genes

Question 17

What do Chip Seq-DNA let us figure out?

Answer 17

GIve data on what DNA sequence proteins bind to

Question 18

Genomic Medicine

Answer 18

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

Question 19

How do we use sequencing in epigenetics?

Answer 19

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

Question 20

Metagenomics

Example?

Answer 20

Studying the sequence of the microbiome

Dutch famine study

Question 21

What gene is vital for metagenomics?

What is special about it?

What can it tell us?

Answer 21

16s rRNA gene

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

Question 22

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

Answer 22

Metagenomics

Question 23

What are the steps for Nucleic Acid Hybridization?

Answer 23

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

Question 24

Southern Blot

Answer 24

Used to identify DNA expression

Question 25

What is photolithography (Affymetrix) still used for?

Answer 25

Genotyping

Question 26

What is the general process for PCR?

Answer 26

Take a pile of DNA, amplify specific sequence, clone it 1. Denature 2. Anneal 3. Extension 4. Repeat

Question 27

What does PCR require to run?

Answer 27

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

Question 28

What is one method of assessing gene expression?

Answer 28

Real-time PCR (Quantitative PCR) Formation of product is continuously monitored

Question 29

*in situ* PCR

Answer 29

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

Question 30

Gene Therapy Draw back?

Answer 30

Transfer of genes into other organism to turn proteins on/off ( - ) Foreign DNA is not introduced into genome--only get short-term expression

Question 31

Transgenic Mice

Answer 31

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

Question 32

What methods are used to get cloned genes into mice? Which is easier?

Answer 32

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

Question 33

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

Answer 33

Produce Chimeric mice Breeding can yield homozygotes

Question 34

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

Answer 34

Knock it out "Gene Knockout"

Question 35

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

Answer 35

Homologous Recombination

Question 36

Cre-Lox System What does this allow for?

Answer 36

Takes advantage of site-specific recombinase to introduce gene deletions, inversions, or translocations ## Footnote **Conditional Mutagenesis**

Question 37

Conditional Mutagenesis

Answer 37

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

Question 38

What does CRISPR/Cas System allow?

Answer 38

Allows Regulation Expression

Question 39

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

Answer 39

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

Question 40

What does RNA Interference give us the ability to do?

Answer 40

Turn off mRNA, thus proteins

Question 41

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

Answer 41

Small Interfering RNA

Question 42

What is the manipulation of an inherent system to block RNA transcription? What can these do? Two drawbacks?

Answer 42

siRNA Turn off genes 1. How do we target specific spots? 2. We have a lot of RNAse, which degrades these

Question 43

siRNA What level do they work at and how?

Answer 43

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