Techniques/applications Of Molec Biology

Front 1

What are restriction enzymes?

Back 1

Bacterial proteins (endonucleases)
DNA binding proteins with nuclease domains - can cleave ds DNA

Front 2

How are restriction enzymes used for bacterial self defense?

Back 2

- Cleave foreign nucleic acids (phage infection, promiscuous plasmids, etc)
-

Front 3

Why wouldnt a bacterium cleave it's own DNA?

Back 3

methylation of endogenous sequence motifs

Front 4

What are restriction enzymes fundamental to?

Back 4

DNA cloning

Front 5

How do you resolve cleaved DNA fragments into DNA bands?

Back 5

gel electrophoresis. Neg charged DNA migrate away from cathode toward anode.

resolved into DNA bands according to their sizes.

Front 6

Where do restriction enzymes cleave?

Back 6

endonucleases
cleave internal phosphodiester bonds

Front 7

What are exonucleases?

Back 7

progressively cleave the 5' or 3' end phosphodiester bonds

(Restriction enzymes are NOT exonucleases)

Front 8

Cleave can result in sticky ends, meaning...

Back 8

5' or 3' overhang ends.

No overhang = blunt end

Front 9

how do you inhibit a restriction enzyme?

Back 9

methylation...restriction enzyme will not cleave methylated DNA

Front 10

What does DNA ligase do?

Back 10

joins dna fragments - forms PDE bonds between 2 separate DNA fragments

Front 11

What does an exonuclease do?

Back 11

clips single stranded overhangs generated by restriction enzymes, for blunt ended joining by ligase

Front 12

what does DNA polymerase Klenow1 do?

Back 12

Fills in overhangs to create blunt ends for ligation, and to incorporate radioactive dNTPs into DNA to produce radioactive probes for Southern and Northern blots.

Front 13

What does polynucleotide kinase do?

Back 13

adds phosphate group to 5' hydroxy terminus of polynucleotides to generate radioactive probes

Front 14

Can two dna fragments, one with 3' overhang, and the other with 5' overhang, be ligated together?

Back 14

no...must be blunt ends

otherwise, must have complimentary 3'/3' or 5'/5' ends

Front 15

What is the amp resistance gene?

Back 15

A way to tell which cells have/have not taken up the plasmid. If they have the plasmid, they have taken up the ampicilin resistence gene. So non-transformed bacterial cells will be killed by ampicilin in the medium

Front 16

How do you generate a recombinanat plasmid?

Back 16

(Insert DNA fragment into a plasmid vector)

- start with circular double stranded plasmid DNA (cloning vector)
- Cleave with restriction nuclease in polylinker cloning site
- covalent linkage of DNA fragment to be cloned..by DNA ligase.
- --> Recombinant DNA

Front 17

How do you generate a recombinanat plasmid?

Back 17

(Insert DNA fragment into a plasmid vector)

- start with circular double stranded plasmid DNA (cloning vector)
- Cleave with restriction nuclease in polylinker cloning site
- covalent linkage of DNA fragment to be cloned..by DNA ligase.
- --> Recombinant DNA

Front 18

Explain amplification of recombinant plasmid in E coli

Back 18

- ds recombinant DNA plasmid introduced into bacterial cell
- cell culture produces hundreds of millions of new bacteria
- many copies of purified recombinant plasmid isolated from lysed bacterial cells

Front 19

What's the purpose of including an ampicilin resistence gene?

Back 19

To allow for selective growth

Front 20

Why is amplification of bacterial cells possible/

Back 20

Bc bacterial cells divide every 20 miins (rather than mammalian cells which divide every 8-24 hours)

Front 21

What does gel electrophoresis do?

Back 21

To confirm that DNA fragment is correctly inserted into the plasmid vector

DNA inserted into cloning site of plasmid vector.
Digestion with restriction enzyme A
Gel electrophoresis (get DNA restriction fragments) - big fragments migrate slower, small ones move faster/further
Evaluate signals: The fragment spanning the cloning site generated by the recombinant plasmid will be larger than the one produced by the vector

Front 22

How do you make recombinant virion with human genomic DNA?

Back 22

Partial digestion of human DNA, cut out replaceable region of bacteriophage DNA with BamH.
Seal bacteriophage/human dna with ligase.
package recombinant dna with in vitro phage assembly system.
--> recombinant virion w/ human genomic DNA

= Cloning in phage vector

Front 23

How do you introduce recombinant DNA into bacterial cell?
How do you get a lot of cloned human gene, using recombinant phage DNA?

Back 23

Recombinant phage vector packaged in phage head proteins to make infectious phage particles.

packaged phage infect bacterial host cells. grow culture 12-15 hours.

Harvest phage particles from lysed bacteria.

Can then purify large quantity of cloned human gene of interest from the recombinant phage DNA.

Front 24

What is BAC vector?

Back 24

circular DNA carrying origin of replication (ORI) for autonomous replciation of the vector.

Also has antibiotic resistence gene for selective growth.

Front 25

What is the genomic DNA library?

Back 25

collection of all the genetic material (DNA) of an organism, cloned in a vector (plasmid, BAC, or YAC)

Front 26

What is a complementary DNA library?

Back 26

collection of the EXPRESSED genes (mRNA) of a eukaryotic organism cloned in a plasmid vector

Front 27

why can cDNAs be cloned in a plasmid vector, but genomic fragments cannot?

Back 27

Because cDNAs are usually a lot shorter (15 kb vs. 100-500)

Front 28

Give 2 reasons why you'd want a genomic library?

Back 28

1. to sequence all the genes of an organism.
2. To compare expression patterns of genes under different conditions or different drug treatments

Front 29

How do you construct a Genomic DNA library in YAC vector?

Back 29

Cleave YAC vector with EcoRI and BamHI.
Insert human DNA in between.

Yeast cell transformation is the clone.

Front 30

How do you construct a cDNA library?

Back 30

from the total mRNAs isolated from tissue, using a reverse transcriptase.

Front 31

How is protein produced from a cloned vector? (expression cloning)

Back 31

Need signals to promote transcription: -10 (Pribnow box) and -35 regions, bacterial promoter, transcription terminator sequence


Signals needed to ensure translation of the mRNA: Shine-Delgarno sequence, translational start and stop codons

Front 32

how do you synthesize proinsulin by bacteria?

Back 32

1. Take mammalian proinsulin mRNA from pancreas
2. Convert to proinsulin cDNA using reverse transcriptase
3. Join cDNA to plasmid
4. Infect e coli with plasmid
5. you get a transformed bacterium with proinsulin

Front 33

What if you want to express a gene in a eukaryotic cell?

Back 33

bacteria cannot do splicing, post-translational modifications, polyadenylation..
Must construct euk expression vectors for efficient expression of gene in eukaryotic host cells.

Front 34

What is transfection?

Back 34

Purified DNAs of expression plasmids are introduced into euk host cells - for functional studies of gene activities, or for getting large amt of the protein products

Front 35

Name 3 methods of transfection

Back 35

1. Electroporation - poking holes in the cell membrane by applying high-voltage electrical field to allow entry of plasmid DNA.
Efficiency: 10-20% of cells are transfected

2. Liposomes - enclosing plasmid DNA in lipid membranes for fusion/uptake into cell membranes
efficiency: 10-20%

3. Recombinant viruses: packaging plasmid DNA into viral particles and infecting the host cells w/ the recombinant virus. Long and hard process!!
efficiency: 50-100%

Front 36

You suspect that a region has enhancer activity. How do you confirm it?

Back 36

insert the human dna spanning the enhancer, linked to a reporter gene, in a euk expression vector.

Make a mutant reporter gene clone with the enhancer gene deleted.

transfect the normal and deletion mutant clones into cultured human mammary cells.

Use assay reporter gene activity to see if deletion causes big decrease in expression of the reporter gene.

Front 37

How was the human genome primarily sequenced?

Back 37

Chain termination method: DNA to be sequenced is denatured to separate the two complementary strands. Mixed with primers, 4 dNTPs, dideoxy NTPs with fluorescent tags, and primers.

The sequence of the newly synthesized, fluorescent DNA molecules after gel electrophoresis is read by an automatic fluorescence detector.

Front 38

What happens in the dideoxy chain termination method?

Back 38

Takes off another OH from C3 (so neither C3 nor C4 have OHs) - thus, prevents strand extension at 3' end.

so incorporation of dideoxyriboNTs blocks further growth of the DNA molecule

These ddNTPs are labeled with fluorescent dyes. DNA sequence read by fluorescent detector.

Front 39

How does PCR (Polymerase Chain Reaction) work?

Back 39

Purpose: to produce lots of DNA product (amplification!)

1. add excess primers, heat to separate strands.
2. Cool to anneal primers to the strands
3. Synthesize new DNA (Heat-stable polymerase)

So: Denature DNA template, anneal primers, synthesize new DNA

- Rapid detection of genetic mutations and pathogens

Front 40

How are RNA genomes of HIV, H1N1 detected by RT-PCR?

Back 40

RNA transcribed into cDNA by reverse transcriptase; PCR amplification

Front 41

How does a Southern blot work?

Back 41

1. DNA (genomic or plasmid) fragmented by restriction enzyme digestion
2. DNA is denatured into single strands by alkali treatment. A specific, labeled single stranded probe is annealed to the single stranded, denatured DNA
3. Visualize the number and sizes of DNA bands generated by the gene w/ autoradiography

applications: RFLP, VNTR, DNA hypersensitivity mapping

Front 42

What's the difference between Northern and Western blot?

Back 42

Northern - to detect RNA
Western - to detect proteins

Western: Uses antibodies instead of probes

Front 43

How do you use Southern blot to locate open enhancer sites?

Back 43

DNA Hypersensitivity mapping
1. Treat cells w/ DNase, which cleaves at all hypersensitive sites on chromatin.
2. DNA is purified and digested with BamH 1 restriction enzyme.
3. DNA is hybridized to a probe
4. Compare bands from DNA digested with just BamH, versus DNA digested with DNase and BamH --> shows location of hypersensitivity site (= enhancer site)

Front 44

What are polymorphisms?

Back 44

Inherited regions of our DNA that can vary from person to person. Most of our DNA is the same though.

Front 45

Give 3 examples of polymorphisms

Back 45

Single base substitutions
Deletions, Insertions (That alter the number of repeats)

Front 46

What can you use to map out polymorphisms?

Back 46

RFLP, VNTR

Front 47

How can you use RFLP for genotyping sickle-cell disease patients

Back 47

1. Cleave patients' DNA samples with Mst II restriction enzyme.
2. In sickle cell pts, there's a bp substitution in the Mst II site, so cannot cleave there.
3. Southern blotting, hybridization to a probe: Homozygous pts should have 1 large band, heterozygous pts should have the large band plus a normal band

Front 48

How can RFLP be used as a paternity test?

Back 48

Father and child should share about 50% of DNA (so will share some bands)

Front 49

What are variable number tandem repeats (VNTR)?

Back 49

Short nucleotide sequences that are organized into clusters of tandem repeats. Scattered at many sites all over chromosome.

Repeat regions can be surrounded by specific restriction enzyme sites so that section of the chromosome can be cleaved.

Front 50

How do you visualize VNTRs?

Back 50

Southern blotting

Front 51

What makes VNTR even more helpful?

Back 51

If you can look at VNTR on multiple chromosomes (ie. was used for identifying the Romanov family)

Front 52

What are transgenic animals?
What is one practical use?

Back 52

Animals that carry integrated human transgenes.
To study human diseases, effects of drug Tx's.

Front 53

How do you generate transgenic mice? (2 methods)

Back 53

1. Select for cells expressing desired gene. Inject transformed embryonic stem cells into inner cell mass. Implant blastocyst in uterus.Test offspring for gene. Can mate heterozygous offspring to get homozygous transgenic strain.

2. Inject desired gene with vector into pronucleus. Implant the fertilized egg in uterus. Test offspring for gene...

Front 54

How do you measure the in vivo effects of transgenic activities?

Back 54

Mouse came from fertilized egg injected w/ recombinant DNA containing human growth hormone.

Measure levels of growth hormone (should be MUCH higer in transgenic mice)

Front 55

How does nuclear transplantation work?

Back 55

Take nucleus from cells from animal to be cloned, put it in an unfertilized egg cell (after removing its own nucleus). Allow cell division so that it turns into an early embryo. Implant in a surrogate mother (reproductive cloning) or transfer early embryo cells to a culture dish (therapeutic cloning)

Front 56

WHat does therapeutic cloning show?

Back 56

That ES cells have the ability to differentiate into all body cell types.

Front 57

Why did Dolly age prematurely?

Back 57

B/c the development clock f the adult donor nucleus was not reset

Front 58

What is iPS (Induced pluripotent stem cells)?

Back 58

From adult somatic cells
If you introduce these 4 genes which encode transcription factors, you can regenerate embryonic stem cells.

Can reset the develpmental clock: erase epigenetic marks. set it back to a pluripotent state, like embryonic stem cells.