IDENTIFICATION - ISOLATION/SEPARATION - CHARACTERIZATION - MANIPULATION

I. Separation techniques:

a. Gel electrophoresis
    -- agarose
    -- polyacrylamide
        - SDS
        - non-denaturing, isoelectric focussing
        - other e.g. for histones, etc.
    -- starch - enzyme activity survives

b. chromatography
    -- paper, etc.
    -- column -- size, charge, affinity,
             hydroxylapatite (binds ds DNA >> ssDNA), etc.

c. centrifugation
    -- buoyant density - CsCl
    -- sucrose

d. differential degradation

II. Characterization: above techniques &

a. biological activity e.g. enzyme activity, ability to participate
   in rxn, etc = BIOASSAY -- STRUCTURE IS FUNCTION !

b.  polypeptides and proteins:  

    - amino acid analysis
    - N-terminal analysis
    - peptide sequencing
    - polypeptide and protein sequencing; S-S bond location
    - analysis of domains, 3o structure

c.  nucleic acids:  base complementarity:

    - DNA sequencing

        -- Maxam & Gilbert: restricted digests
        -- Sanger: dideoxynucleotide premature termination of replication
            ddNTPs

    - melt -- re-anneal - hybridize

        DNA-DNA duplex; DNA-RNA heteroduplex
        - compare sequences
        - in situ hybridization on chromosome

CoT: CoT1/2 - relates to size

Euk DNA shows triphasic curve: - relates to copy number & size

highly repetitive:
middle  " " :
"unique" = "single copy" = "low copy number:

can compare between genomes: how similar are DNA sequences - what % anneal, how long does it take, etc.

-- use ability to hybridize for isolation (affinity chrom.)

-- & identification:

I.D. nucleic acids or proteins on gels:

BLOTTING: nitrocellulose paper:  "dot blots" for quantitation;
    hybrid selection for isolation, or blotting gels for identification

Southern blot: DNA separated on gel blotted onto paper, DNA probe

Northern blot: RNA separated on gel blotted onto paper, DNA probe

Western blot: protein separated on gel blotted onto paper, antibody probe

STRINGENCY: on filters or on gel blots
    - low stringency for finding non-identical but related sequences
      e.g. analogous or homologous genes in other species

How to label a probe:

NICK TRANSLATION: DNA + DNAase I + DNA pol I + *NTPs
    where DNase I nicks the DNA, DNA pol I repairs with labeled NTPs, but N.B. that repair is not one but several nucleotides long using 5'- 3' exonuclease activity of Pol I, so now DNA has radioactive patches through its middle.

    boil (melt DNA), then cool quickly to prevent re-annealing = probe

How to find or make a probe:

cDNA - REVERSE TRANSCRIPTASE

-----------------------------------AAA (even if terminal transferase has to be used)    + oligo (dT)

-----------------------------------AAA         reverse transcription
        <------------------TTT

---------------------------------AAA         reverse transcription
/--------------------------------TTT
\------        alkaline hydrolysis to degrade the RNA -->

/---------------------------------TTT
\------      Now use DNA polI  to complete the strand


/---------------------------------TTT
\---------------------------------AAA        and nuclease S1

----------------------------------TTT 5'        to clip the ssDNA
----------------------------------AAA 3'

Use synthetic linker or "polylinker" - 8-12bp - to create desired ends



CLONING DNA:

    VECTORS:

1. small, well-characterized DNA molecule
2. has origin of replication "ori"
3. has ability to survive & replicate autonomously
4. can be retrieved; it can be distinguished from host genomic DNA
5. usually want several UNIQUE RESTRICTION SITES to increase efficiency
    of manipulation - knowing exactly what each restriction enzyme does
5. SELECTION: has 1, usually 2 or more selectable markers
6. sometimes want facility to EXPRESS DNA of interest = EXPRESSION VECTOR
7. sometimes want to be able to grow in bacteria (rapid growth &
    accumulation) then express in eukaryote without further DNA cutting & pasting ... SHUTTLE VECTOR = has 2 set of criteria # 2,3,4,5,6

EXAMPLES:

1. PLASMID: small circular DNA with ori

    one popular one is bBR322 for E. coli which has 2 antibiotic resistance genes for selection.  Other selection devices include nutritional supplement requirement, color rxn resulting in a noticeable color from some odd-ball rxn product (or the known product of a gene being stalked: use mutants with requirement for a particular supplement.  Transform with plasmid DNA which rescues the auxotrophy.  Then, look for responsible gene on plasmid, and only later genomic DNA.

RESTRICTION ENDONUCLEASES: remember lab. Sticky vs blunt ends - Use for:

        -- terminal transferase to build desired sticky end
        -- cloning sites pre-made
        -- ligation

    - mapping - generate library or bank, or direct restriction mapping
      as in lab

    - RFLPs: these are a PHENOTYPE that can help identify a GENOTYPE

    - manipulation

MAPPING SEQUENCES:

    Use restriction endonucleases or random shearing to generate a

LIBRARY or BANK: fragments containing -hopefully - all of the organism's genome in convenient size pieces.  These are cloned, each into a single vector, and each vector into a single host, and used en masse for screening.  A library can be set up in any of several ways:

    A. LIMIT SEQUENCE POPULATION:

-- whole genome or partial e.g. chromosome, even part of a chromosome
    
-- cDNA representing the EXPRESSED portion of the genome under particular circumstances

    B. CHOOSE VECTOR:

-- BY SIZE:

a. plasmid: small, may be high copy number, easy to work with
    e.g. 20-50X/cell, and using antibiotics which affect host but not plasmid replication, up to
         about 1000X/cell.
    easy selection mechanisms, easy to manipulate
    holds 5-10kbp

b. phage lambda: can accommodate larger pieces of DNA - 15-kbp
    total length restricted to 45kbp; need to remove unnec. lambda seqs

c. other viruses may be used but are not so popular

d. cosmid: take genes for packing = "cos" + ori + genes for selection, resistance gene   total about 5 kbp  - so now this can accommodate inserts of about 30-40kbp
 -- usually for genomic banks (lambda cosmid or phagemid).

e. single stranded phage such as M13: use cDNA without making it double stranded; convenient for sequencing by one of the methods.

f. EXPRESSION VECTORS: can look for sequences (esp. cDNAs) capable of supporting expression, so here, the probe might be a bioassay, or an antibody!

    These have easy-access promoters next to insertion site, and may have regulatable promoters.

    Some expression vectors are set up to test promoters & enhancers -- they have in them a REPORTER GENE whose product is readily QUANTITATED so that efficiency can be directly assessed.  BLUGENE - ß-gal makes product that turns blue.

-- SHUTTLE VECTORS: allow replication to high copy number in prok. system, then transfer without further manipulation into eukaryotic system for expression. -- even into mammalian cells for complex regulation & processing or even gene therapy.

STRATEGIES:  shotgunning to make a bank, then selection from the library

-- or find a specific sequence - "fish" it out using any set of tricks outlined above, and clone it     straight.

    There are now more ways to do this, e.g. synthetic oligonucleotides whose sequences are
 inferred from polypeptide seq. - N.B. problems with ambiguity!  and PCR - later.

Detection might then be Southern, or Western if an expression vector, or other manipulation is used.

CHROMOSOME WALKING

SITE-DIRECTED MUTAGENESIS

PCR

CHIPS: analysis of
    - genome
    - expressome
    - proteome
    - etc. !