Chapter 15: Recombinant DNA

Recombinant DNA: new combination of DNA segments or DNA molecules not found together naturally

restriction
enzymes:- isolated from bacteria, name based on source species (EcoRI= E. coli)

- used to cut DNA molecules at specific sequence or into specific sized pieces

- types I (nonspecific cutting sites) and II (specific cutting sites)

- type II recognition sequences are symmetrical, eg, palindromes (sequences that read the same in both directions; 5'-3' on leading strand: GAATTC and CTTAAG on nonleading strand, 3'-5'); Fig 15.1

- can produce sticky (overhang on one strand) or blunt (no over hang) ends; Fig 15.2

vectors:- cloning vehicles which can gain entry into host cell and be replicated or cloned; three main types: plasmids,bacteriophages, cosmids

- can replicate autonomously in host cells

- contain a large number of restriction enzyme cleavage sites that are present only once with vector

- contain selectable marker to assess if vector has been inserted into host

clones:- identical copies of target DNA inserted in vector are amplified within a host cell

host cell: - as it replicates it passes the vector to its progeny; eg, bacteria: E coli, eukaryotes: yeast, (2-micron plasmid is used as vector); artificial yeast chromosomes (YACs can hold DNA segments 1 Mb long -- used for mapping)

Vectors /traits	plasmid: circular, extrachromosomal DNA molecule; infectious agent	bacteriophage; virus that infects bacteria	cosmid
	naturally occurring, extra-chromosomal, site of origin, replicate autonomously	naturally occurring, site of origin, replicate autonomously	hybrid vectors; lambda & plasmid DNA, site of origin,replicate autonomously
example	pBR322	lambda	cos(lambda) mid (plasmids)
size vector
size insert	5-10 kb	central 1/3 dispensable and can therefore be replaced; 15 kb	50 kb
selectable marker	resistance to ampicillin		resistance to ampicillin
selectable marker	resistance to tetracycline		resistance to tetracycline
replication potential	replicates to produce ~10-50 copies/cell
example	pUC18	M13	shuttle vectors
size vector	1/2 size of pBR322
size insert	larger than pBR322
marker	lacZ gene, vector incorporated indicted by blue colonies on plates with X-gal; insertion of target DNA indicated by white colonies
resistance to tetracycline
replication potential	replicates to produce ~500 copies/cell

Cloning- E coli (Fig 15.14)

1. target DNA cut with appropriate restriction enzymes
2. vector cut with same restriction enzyme (or enzyme producing complimentary 'ends'

inserting target DNA
sticky ends: need DNA and vector cut with same restriction enzyme, sticky ends will match up (H bonding), fragments bound using DNA ligase
blunt ends: join molecules using deoxynucleotidyl transferase to add poly T ends to one source and poly A to other, tails line up and DNA ligase closes gaps.

3. fragments ligated to plasmid molecules creating recombinant vector
4. recombinant vector transferred into bacterial host cells (usually by transformation)

Introduction of lambda into host: transection or transformation:

- host cells made permeable chemically and then mixed with ligated molecules

- vector molecules taken into host cells where they direct the synthesis of the infective phage

5. host cells are grown on nutrient plate to form colonies (cells within given colony are clones)
6. colonies that have taken up the vector are identified

Selected Marker
vector present: host cells are plated onto plates with antibiotics (eg, tetracycline) -- if vector which is antibiotic resistent has been incorporated then host cells will grow

7. colonies with vectors that contain target DNA are identified and harvested

target DNA present: incorporation will have interrupted 2nd marker (eg, ampicillin); use replicate plating to ID colonies: press plate onto second plate with 2nd marker antibiotic present; colonies which DON'T GROW have the target DNA incorporated. Compare two plates to ID colonies that didn't grow the second time.

Other Applications:

Constructing Libraries
Mapping DNA/genes

cDNA: eukaryotes fig 15.19

- use mRNA as template, -
primer is poly T which lines up with poly A tail, -
reverse transcriptase produces complementary DNA strand-
RNA strand removed with ribonuclease H-
ssDNA then duplicated with DNA polymerase I, 3' end of template DNA tends to fold back into hairloop and so acts the primer
- S1 nuclease removes hairloop, producing regular ds DNA, complementary or cDNA which can now be cloned

contains only expressed sequences present in at given development stage or type of cell