FUNDAMENTALS OF BIOTECHNOLOGY

• Treatment with R.E produces sticky ends after ligation with target DNA.. • Blunt ends will bind to the blunt ends of target DNA to produce new DNA with sticky ends.. • Immunological sc

FUNDAMENTALS OF BIOTECHNOLOGY

Lecture # 08

• Linkers & Adaptors

• Selection and Characterization of rDNA

• cDNA Libraries

• DNA transformation

Linkers and Adaptors

• Sticky ends are desirable for DNA cloning

experiments

• Provided by treating the target and vectors with same R.E or with different but producing the

sticky end

• But some time target DNA blunt ended

• So therefore we will have to use Linkers and

Adaptors

• Synthetic , Short and known double stranded

oligonucleotides sequence

• Having blunted ends on both sides and R Sits.

• Treatment with R.E produces sticky ends after ligation with target DNA.

• e.g Linker having sit for BamHI

• Drawback if target DNA also having the same R Site then?

•

(Please use the book notes for detail)

• A Synthetic dstranded Oligonucleotide having

blunt end and Sticky end

• Blunt ends will bind to the blunt ends of target

DNA to produce new DNA with sticky ends

• Problems: sticky of adaptors will binds with each other so…

• Treatment with Alkaline Phosphates

• After attachment with target…… treatment

• Polynucleotide Kinase to add P–OH at 5 prime

• e.g Complimentary poly (C) and poly (G) for

vector and target DNA respectively

Selection and

Characterization of rDNA

Selection and Characterization of rDNA

• Identification and selection of rVector:

• Desirable antibiotic resistance

e.g Ampicillline and tetracycline

• plaque formation:

X-gal

• Next step is to know which one contains our DNA of

interest.

• Genetic Method (Resistance, Color, plaque formation).

• Immunological screening: (inserted gene will produce protein Ags and Abs reaction based)

• Hybridization method:

Prob will be hybridized with the corresponding DNA.

• DNA Sequencing:

• Southern and northern hybridization analysis

• Western blot for protein analysis

Screening and characterization of rClones

Selection with antibiotic resistance (ampr)

Twin antibiotic resistance

Blue-white screening

Restriction digestion and Gel Electrophoresis

Hybridization to identify the interested

DNA or its RNA product

1 Radiolabeled probes which is complementary to

a region of the interested gene

Probes :

 An oligonucleotide derived from the sequence

of a protein product of the gene

 A DNA fragment/oligo from a related gene of

another species

1 Blotting the DNA or RNA on a membrane

2 Hybridize the labeled probe with DNA

membrane (Southern) or RNA (Northern)

membrane

Screening by Hybridization

Probes: DNA or RNA 100+ bp in size good Sequence match >80% best Stringency conditions

Screening Colonies by Hybridization

• Nucleic acid probe

• Cells transferred to nylon membrane and lysed

• DNA binds to membrane, is denatured and probe hybridized

• Bound probe detected

by autoradiography after washing membrane

Screening by Immunological Assay

Screening by Functional Complementation

• Requires strain unable to produce desired

Southern and Northern blotting

Western blotting using a specific antibody

Identify the protein product of an interested

gene

PCR (polymerase chain reaction)

to amplify a sequence of DNA using a pair of primers each complementary to one ends of the target DNA sequence.

Running on Gel separation size based.

Nucleic acid sequencing

DNA sequencing

Two main methods:

Maxam and Gilbert chemical method

the end-labeled DNA is subjected to basespecific cleavage reactions prior to gel separation.

Sanger’s enzymic method

the latter uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer.

DNA libraries

 DNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by

propagation in the host

 A collection of DNA fragments representing the genome of an organism inserted in a

suitable vector

 A clone is a genetically distinct individual or set

of identical individuals

DNA libraries

Genomic libraries :

Genomic DNA library represents total genomic DNA

prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the

non-coding DNA

cDNA libraries :

cDNA library represents expressed genes (reverse transcribed mRNA)

DNA copies (cDNA) synthesized from the mRNA by reverse

transcription are inserted into a vector to form a cDNA library

DNA libraries

Large genomes need large numbers of clones

to achieve high coverage

Number of colonies needed for 95 % coverage:

Species Genome size (bp) number of clones (35 kb)

To reduce clone numbers in a library: increase

insert size

Isolation of Poly(adenylated) mRNAs

Matrix

• S1 nuclease

 Degrades ss nucleic acids (unpaired

loop)

Enriching for Full Length cDNAs (1)

• Primer has adapter (RE cutting sequence)

• Ribose ends of mRNA are biotinylated

• RNase I degrades ss RNA

• Only full length cDNA is still attached to a biotinylated mRNA (biotin still on 5’end)

• Capture full length copies

adapter

Enriching for Full Length cDNAs (2)

When to use genomic or cDNA libraries

cDNA library:

when you want to express a protein

when you want a tissue specific library

Genomic DNA library:

when you want to study non-coding sequence:

intron/exon structure promoter

centromeres/telomeres replication origins

unexpressed (or very lowly expressed) genes