Biochem 8/25

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DNA cloning

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separating specific gene or DNA segment from larger chromosome, attaching it to small molecule of carrier DNA, and replicating it many times

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Cloning entails 5 general procedures

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1)Cutting DNA at precise locations (sequence-specific restriction endonucleases)

2)A small molecule capable of self-replication (vector)

3) Joining two DNA fragments covalently (DNA ligase does this)

4) Moving recombinant DNA from test tube to a host cell that will replicate it

5) Selecting or iding host cells that contain recombinant DNA

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Which type of restriction endonuclease cleave DNAs at specific base sequences, within the recognition site?

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Type 2

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Polynucleotide kinase

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adds phosphate to 5' )H end of polynucleotide to label it or permit ligation

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Terminal transferase

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Adds homopolymer (ex GGGGGG) tails to 3' OH ends of linear duplex

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Alkaline phosphatase

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Removes terminal phosphates from either 5' or 3' end

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Polylinker

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synthetic DNA fragment containing recognition sequences for several restriction endonucleases that can be inserted into a plasmid

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Two methods used to produce target DNA suitable for cloning

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1) Genomic DNA: each clone contains a fragment of the entire genome of the organism
2) Complementary DNA (cDNA)

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Genomic Library: How does it work?

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-Collection of all DNA needed to read everything about the genome

-isolated from cell nuclei
(any cell)

-digested with restriction enzymes

-fragments ligated into lambda arms

-phage particles are assembled with packaging proteins

-lyse phages in alkaline solution to release DNA

-Hybridize with labeled probe

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Contig

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set of overlapping DNA segments derived from a single genetic source. A contig in this sense can be used to deduce the original DNA sequence of the source

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cDNA libraries

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these will ONLY be representative of mRNA sequence (no introns)

will be specific for the type of cell you're studying

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cDNA production and cloning

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Start with blunt ended, ss mRNA.

- mRNA has poly A tail which is useful. Make a bunch of Ts to hybridize with

Now use <b> reverse transcriptase </b> to fill in the dNTPs

Leads to a RNA/cDNA hybrid.

Now remove RNA with alkali (DNA doesn't get destroyed as easily by alkali)

Add poly dG tail (using terminal transferase)

DNA polymerase is used to produce complementary strand to cDNA

Add methyl group to prevent cleavage

Ligate ends of cDNA to restriction site linker which is a recognition sequence.

Add restriction enzyme --> cleavage

Put into phage, infect E. coli

cDNA libraries are used to express eukaryotic genes in prokaryotes

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What 3 properties must a cloning vector posses to function in DNA cloning?

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1) presence of restriction site (allow insertion of DNA)

2) autonomous replication in a host cell, permitting amplification of target DNA independently of host cell DNA synthesis

3)Selectable feature that permits transfected cells to be distinguished from cells not containing the vector

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How big can target DNA be to fit into a plasmid vector?

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Up to 5 kilobases (kb)

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How big can target DNA be to fit into a lambda phage vector?

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up to 20 kb

also it takes up the DNA fragments more easily

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How big can target DNA be to fit into a yeast artificial chromosome?

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1000kb

These have been very useful to study human genes which are long because of introns

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Southern blotting

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Technique to detect a specific DNA sequence

Detects DNA with labeled DNA probe

Useful to ID a specific restriction fragment out of millions present in restriction digest of individual's genomic DNA

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Northern blotting

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Detects RNA with labeled DNA probe

Useful in determining whether a specific mRNA is expressed in a particular tissue

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Western blot

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Detects present of protein with a labeled antibody

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PCR: basic definition

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in vitro enzymatic method for amplifying a specific sequence of DNA located between 2 short flanking sites whose sequences are known

Requires that at least a portion of the sequence be known so primers can be made.

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For PCR, what is needed in addition to the DNA you want to amplify?

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1) Single strand primers (15-25 bp) complementary to flanking sites

2) DNA polymerase that will NOT be ruined by heat (from thermophile)

3) All four dNTPs

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PCR: describe events

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1) Separate strands with heat

2) Cool and add primers

3) Add dNTPs and heat-stable DNA pol

4)Heat to denature, then cool (with primers, dNTPs and DNA pol present)

5) continue

Eventually strands containing DNA other than that of interest (which started with the primers) get diluted out, and only the DNA of interest remains

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Restriction fragment length polymorphisms

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DNA polymorphisms are small heritable variations in DNA found in general population, occurring every 200-500 nts, but most cause no phenotypic effect

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What does restriction mapping entail?

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IDs the restriction sites in a region of DNA and distance in kb between them, involves digesting samples of a cloned DNA sequence with different restriction enzymes, detecting resulting restriction fragments by southern blotting, and comparing their lengths

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What are some uses for RFLP analysis?

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DNA fingerprinting

detecting certain genetic diseases