Genetics Exam Iii: Recombinant Dna

Front 1

Enzymes that cut DNA only a specific sequences

Back 1

Restriction endonucleases (enzymes)
Sequence-specific endonucleases

Ie: EcoRI & BamHI

Front 2

Restriction enzymes have what type of symmetry?

Back 2

Dyad symmetry (not really a palindrome) but if you write both strands and read from the 5'end get same thing.

Front 3

What are the functions of restriction enzymes

Back 3

1. Biological-natural bacterial defense mechansims (cut up foreign DNA)
2. Technological-very accurate/precise "DNA scissors"

Front 4

Function is join thephosphoribose backbones

Back 4

DNA Ligases

Requires free 3'OH end next to free 5'-PO4

Front 5

How is recombinate DNA formed?

Back 5

Cut 2 types of DNA with same restriction endonuclease-->ends compatible-->mix DNA together-->seal with DNA ligase

Front 6

A DNA molecule consisting of components from different "parent" molecules

Back 6

Recombinant DNA

Front 7

Making many copies of the recombinant DNA using host organism

Back 7

Cloning

Front 8

DNA molecule that can be replicated

Origin of replication has to be specific to a particulart host organism

Back 8

Vector

Front 9

DNA fragment from organism/individual of interest, which will be joined to a vector

Back 9

Insert

Front 10

The process by which the recombinant DNA is put into the host

Back 10

Transformation

Front 11

Essentials of vectors

Back 11

1. Insertion site: must have endonuclease
2. Must have an origin of replication that matches host
3. Selectable marker-for screening

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Types of vectors

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1. Plasmid vectors
2. Lambda-phage
3. Cosmids
4. Artificial chrom.

Front 13

Insert size range of the types of vectors

Back 13

1. Plasmids: up to 10kbp
2. Lambda-phage: 10-15 kbp
3. Cosmids: 25-40 kbp
4. BAC: 50-200 kbp
5. YAC: 200-1000 kbp

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What are the 2 classes of inserts

Back 14

1. Genomic DNA insert
2. cDNA insert

Front 15

What do we get when we chop up our DNA from an organism?

Back 15

W/e info is in the genome
1. Protein coding regions (exons)
2. Introns
3. Regulatory info (promoters, enhancers, silencers)
4. Sapcer DNA-repetitive DNA "junk"

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How to make cDNA

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1. Isolate mRNA by using poly T beads that trap poly A tails
2. Synthesize cDNA using reverse transcriptase
3. cDNA now inserts to be attached to vectors.

Front 17

What is the information content of cDNA clones?

Back 17

1. Same info as mRNA: ORF, untranslated regions, poly A tails
2. Will not have introns, promoters, enhancers, silencers, spacer DNA

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Expression vector

Back 18

To get host to make protein encoded by cDNA, requires expression vectors.

Front 19

What is needed for an expression vector

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1. Origin of replication
2. Prokaryotic promoter
3. Prokaryotic ribosome binding site
4. BACTERIAL RNA polymerase
5. BACTERIAL ribosome

Front 20

Are there introns in the cDNA copy that is inserted into the expression vector?

Back 20

No, introns were already spliced out since these are proteins.

Front 21

Clone libraries

Types

Back 21

A collection of clones, made simultaneously, from a given sample.

1. Genomic libraries
2. cDNA libraries
3. Expression libraries

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Genomic libraries

Back 22

Use recombinant DNA with genomic DNA inserts

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cDNA libraries

Back 23

Use recombinant DNAs with cDNA inserts

Front 24

Expression libraries

Back 24

Use recombinant DNAs with cDNA inserts ligated to expression vectors

Front 25

Identification of recombinant clones. Need to distinguish:

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1. bacteria containing no plasmids at all.
2. Bacteria containing plasmids with no inserts
3. Bacteria containing plasmids with some inserts
4. Bacteria containing plasmids with desired inserts

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Biological selection

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Only the bacteria with the vector survive.

Plate antibiotic and only those that picked up vector will survive.

Front 27

Screening

Back 27

Sort through all of the survivng bacteria (with the vectors) to find the bacterial colony with the DNA insert of interest.

Front 28

Molecular probes

Back 28

To find similar sequence of expressed protein of interest.

Front 29

Nucleic acid probes (for genomic DNA libraries and cDNA libararies)

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DNA, RNA, synthetic oligonucleotide that must be labeled with either radioactive or fluorescent label.

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Antibody probes (for expression libraries)

Back 30

Ab binds at sites where protein being made.

Front 31

How is the probes used?

Back 31

1. Plate bacteria
2. Use a filter and place on plate
3. Lyse the cells on the filter NOT the plate.
4. Denature DNA which fixes itself to the filter
5. Add labeled probe
6. Find your desired colonies (usually with x-ray film)
7. Overlay filter on plate and so know our colonies that contain the inset.

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Goals of molecular analysis

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1. Identify gene that contains mutation causing specific phenotype
2. Identification of the mutation that causes specific phenotype
3. Identify individuals bearing mutation that causes phenotype
4. Determine size and amount of the mRNA or protein expressed from a given gene.

Front 33

Southern blot analysis

Back 33

Detecting specific DNA fragments
1. use restriction enzymes to get fragments
2. separate DNA fragments by size using gel electrophoresis, smallest fragments travel towards bottom and largest stay towards top.
3. Denature DNA in gel
4. Transfer DNA to filter
5. Hybridize to nucleic acid probes
6. Expose to x-ray films. Binds appear indicating position of hybridized fragments.

Front 34

What is the result of Southern blot analysis?

Back 34

Bands on film indicate SIZE of DNA fragments with the sequence of interest.

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Northern Blot

Back 35

Detecting size and abundance of RNA species

1. Isolate RNA
2. Separate RNA's by size using electrophoresis
3. Transfer to filter
4. Hybridize to nuclei acid probe to find our RNA fragments of interest

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The result of Northern blot tells us?

Back 36

Position of bands tells us SIZE of RNA of interest

Intensity of band tells us AMOUNT of RNA is in this sample.

Front 37

Western blot

Back 37

Detecting size and abundance of PROTEIN species

1. Isolate proteins from tissue
2. Separate by size using electrophoresis
3. Transfer to filter
4. Use antibodies to detect specifiic proteins of interest

Front 38

What does is the result of the Western blot?

Back 38

1. Position of band on filter tells us SIZE of protein

2. Intensity of band tells us AMOUNT of protein on tissue

Front 39

PCR Polymerase Chain Reaction

Back 39

Amplication of a specific DNA fragment

Do not need a vector or an origin if replication. But DO need a gene-specific primer

Front 40

Gene-specific primers for PCR

Back 40

1. Are short ssDNA molecules
2. DNA polymerases need free 3'OH end to attach incoming nucleotides.

Front 41

process of PCR

Back 41

1. Denature dsDNA to get single stranded
2. Anneal primers
3. DNA synthesis using DNA pol that is heat stable

Front 42

What type of growth is seen in PCR?

Back 42

Exponential increase

Each PCR cycle doubles the amount

Very fast.