Ch 1. My Q's Flashcards

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1
Q

Largest stretch that can be sequence?

A

600-800bp

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2
Q

How did they increase landmark density in maps?

A

development of other variable molecular markers (not just RFLPs)

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3
Q

Solutions to the human genome project

A

ave milestones (genetic map, physical map), sequence model organisms for comparison, develop strategies (automated, computer databases)

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4
Q

Why isolate single genes?

A

Learn from sequence (is it normal?), Modify genes to study function (knockouts), Move genes between organisms (transgenics)

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5
Q

What’s cDNA

A

complementary DNA synthesized from single stand RNA (reverse transcription

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6
Q

How can we isolate a gene from within DNA

A

Extract total DNA (or cDNA) from organism under study —> Cut into pieces (restriction enzymes -RE) –> Isolate a vector and open it up using RE –> Insert DNA pieces into the vector (Recombinant DNA). –> Insert in a cell system (e.g. bacteria) that produces a colony of genetically identical progeny (clones). –> Identify clone carrying the DNA piece of interest

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7
Q

What are Restriction enzymes?

A

Enzymes that recognize and cut specific 4 to 6 palindrome sequences of DNA. Fragments with sticky ends can be ligated to vectors.

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8
Q

3 things to make a cloning vector

A

origin of replication (to amplify donor DNA), selectable marker, At least one unique restriction enzyme cleavage site

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9
Q

Plasmid

A

small circle double stand (dsDNA), ORI, some antibiotic resistance maker, restriction sites

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10
Q

How is vendor and donor DNA sealed?

A

DNA ligase Phosphodiester bonds at junctions It can also be used on blunt ends

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11
Q

Selection of recombinant clones

A

Use RE to cut the plasmid and the DNA, (plasmid at TetR) —>ligate the two together—> grow on ampicillin which both have resistance too—> make a stamp of each plate and regrow, one on amp and one on tet. Only non-recmombinate will grow on txt because no foreign DNA.

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12
Q

3 other vectors

A

Viruses (infect cells with high efficiency. Can take fragments longer than 10Kb. Remember that some eukaryotic genes can be quite long. ) Cosmids (can carry up to 45Kb long donor DNA. They can replicate as plasmids and can be packaged into phage heads. ) Artificial chromosomes ((BACs, YACs) can carry longer fragments of DNA (cloning full genomes).)

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13
Q

What’s a DNA library

A

A collection of clones, each containing a different sample of the donor DNA of interest.

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14
Q

How to use probe

A

Prove is a piece of DNA that searches for a sequence within the clone. Plasmid vector—>Trasform Ecoli cells—>velvetten stamp—>Filter removed. Bacteria lysed. DNA denatured and bound to filter—>labeled pride solution added—>Detect hybridization by autoradiography or radiolabeled prove and chemiluminescent election for non radiolabled probe. Dark spot=clones detected by probe

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15
Q

Using cloning to isolate disease-alleles

A

The disease is inherited together with an allele of a random molecular marker very often if RMM is close by. Ex: Cistic Fibrosis is found to intagrate with 2 MM (MET - D7S8) 98% of the time therefore it’s within he 2.5Mb distance between said markers.

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