CH7.1 – DNA Cloning & Technique Requirements

Chapter 7 focuses on DNA cloning and the technical requirements for studying genes and their products.
Central problems addressed:
- Genomes are huge (e.g., human genome ≈ $3 \times 10^{9}$ bp) → difficult to analyze a single gene without isolation.
- Cloning allows targeted isolation, amplification and functional study of specific DNA segments.

Enables detailed analysis of one gene among millions.
Provides abundant, identical copies for sequencing, mutagenesis, expression analysis or protein production.
Permits storage of genetic information in libraries for future retrieval.

Select a cloning vector (yellow plasmid in schematic).
Isolate DNA of interest from total genomic DNA (red segment in schematic).
Join DNA fragment with vector → forms a recombinant vector (recombination = DNA from ≥2 sources combined).
Introduce recombinant vector into a host cell (transformation, e.g., E. coli). The host simultaneously retains its own chromosome.
Apply selection to recover only cells harboring the recombinant vector (antibiotic resistance, metabolic markers, etc.).

Origin of replication (ori)
- Distinct from chromosomal origin; ensures autonomous replication.
- Species-specific sequence; without it the vector is lost after few divisions.
Selectable marker(s)
- Usually antibiotic-resistance genes so that only transformed cells grow.
- Must remain functional unless purposely disrupted for screening.
Unique cloning site(s) (multiple cloning site, MCS)
- Single occurrences of restriction enzyme recognition sequences.
- Prevents unintended large deletions if site appears twice.

Circular, medium-copy plasmid.
Features:
- Ori (propagates in E. coli).
- bla (Amp^R) – β-lactamase; resistance to ampicillin.
- tetR (Tet^R) – resistance to tetracycline.
- MCS containing Pst I, Eco RI, Bam HI, Sal I sites (only one of each).

BAC (Bacterial Artificial Chromosome)
- Insert size ≈ $3 \times 10^{5}$ bp (≈300 kb).
- Ori for E. coli.
- cat gene → chloramphenicol resistance (selectable marker).
- MCS embedded in lacZ → enables blue/white screening (extra layer of selection, discussed in class).
- Low-copy (1–2/cell) → carries par genes (partition genes) guaranteeing segregation during cell division.
YAC (Yeast Artificial Chromosome)
- Insert size up to $2 \times 10^{6}$ bp (≈2000 kb).
- Two ori sequences → replicates in E. coli (plasmid form) and yeast (chromosomal form). Hence a shuttle vector.
- Yeast-specific components:
- CEN (centromere) sequence → attachment to mitotic spindle.
- TEL (telomeres) at both ends once vector is linearized (protects ends from degradation/shortening).
- Yeast selectable markers (e.g., URA3, HIS3, etc.).
- Cloning site (Eco RI) positioned so removal of an intervening segment yields a linear chromosome with TEL–insert–TEL architecture.

Restriction enzymes recognize short palindromic DNA sequences and introduce double-stranded breaks.
- Example recognition sites:
- Bam HI: $\text{G}\,\text{GATCC}$
- Pst I: $\text{CTG}\,\text{CAG}$
Cleavage patterns
- Eco RI → staggered cut, 5′ overhangs ("sticky ends").
- Pvu II → blunt cut (no overhangs).
Experimental workflow:
1. Digest genomic DNA and vector with compatible enzyme(s).
2. Purify desired fragment (gel extraction or size selection).

Sticky ends from compatible enzymes anneal via base pairing.
DNA ligase (ATP-dependent) seals nicks, forming phosphodiester bonds between 5′-phosphate and 3′-OH ends.
Works with either sticky or blunt ends (sticky more efficient).

Chemical (CaCl₂) heat-shock
- Cells kept ice-cold → brief 42 °C pulse → membrane becomes transiently permeable.
Electroporation
- Electrocompetent cells given short, high-voltage pulse.
- Generates pores → higher efficiency than heat-shock.

Transformation is never 100 % efficient; mixture of:
- Cells with no vector.
- Cells with non-recombinant vector.
- Cells with recombinant (desired) vector.
pBR322 double-antibiotic screening example
1. Insert cloned into Pst I site inside bla → disrupts Amp^R.
2. Plate on tetracycline → selects any cell carrying pBR322 (Tet^R still intact).
3. Replica-plate surviving colonies onto Tet+Amp plates.
- Grow = Tet^R Amp^R → no insert (vector intact).
- No growth = Tet^R Amp^S → desired recombinant (bla disrupted by insert).
1. Pick corresponding colony from original Tet plate for further work.

Genomic DNA digested with a restriction enzyme → millions of fragments.
Each fragment ligated into vector → transformed into host.
Ensemble of colonies covers entire genome; each clone harbors a different genomic insert.
Useful for whole-genome sequencing, physical mapping, promoter analysis.

Represents only expressed genes (no introns or intergenic regions).
Procedure:
1. Isolate RNA (often poly-A mRNA).
2. Reverse transcriptase synthesizes first-strand cDNA using RNA as template.
3. DNA polymerase synthesizes second strand.
4. Clone double-stranded cDNA into vector.
Applications: expression profiling, isolation of coding sequences, production of recombinant proteins without introns.

Recombinant DNA – artificial assembly of DNA from different origins.
Sticky ends – overhanging single-stranded termini enabling complementary base pairing.
Blunt ends – flush ends; no overhang.
Low-copy plasmid – maintained at ≤2 copies per cell; requires partitioning system (par genes).
Shuttle vector – replicates in two distinct host species.
Transformation – introduction of exogenous DNA into prokaryotic cells.
Selection vs. Screening
- Selection: condition kills cells lacking a feature (e.g., antibiotic resistance).
- Screening: differentiates phenotypes without necessarily killing (e.g., blue/white).

The combination of unique restriction sites and selectable markers makes cloning precise and traceable.
Vector choice is dictated by insert size, host organism, copy-number needs and downstream applications.
Libraries broaden cloning from "one gene at a time" to genome-wide or transcriptome-wide studies.
Mastery of cloning techniques underpins modern genomics, biotechnology, and therapeutic protein production.