ICSD PART 1

Restriction enzymes:
Natural functions: Fragment the viral DNA leaving its own intact: bacteria have enzyme methylases: add CH3 to its own DNA, once reconition sequence on its own DNA is methylated, RE cannot recognize the sites.

Properties:
Highly specific Palindromic: identical when read forwards or backwards on complementary strands Have sticky/blunt ends after digestion

Use of Restriction enzymes/sites for the formation of recombinant DNA molecule

After being cut with RE, blunt ends/sticky ends form, sticky ends preferred as H-bonding b/w base pairs can occur. (Temporary associations) higher chance of 2 restriction fragments being annealed with DNA ligase catalyses the formation of phosphodiester bonds b/w nucleotides.

Procedures to clone a eukaryotic gene in a bacterial plasmid

Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate, i.e. virus, bacterial plasmid

Characteristics of an ideal cloning vector:

1. Origin of replication: so that DNA of interest can propagate many copies of DNA of interest along with cloning vector. 2. Selection markers: confer a well-defined phenotype on host organism helps to identify the host cell in which the foreign DNA can be found. 3. Multiple cloning sites: DNA sequence containing SEVERAL RE sites at which DNA fragment can be inserted in. NOTE: these unique sequences only occur ONCE through the vector. insertion of gene at these sites cause disruption of a gene that codes for a phenotype

Cloning procedures
1. Isolation PCR (short sequence), RE digestion (flanking), mRNA: cDNA can be synthesized (Reverse transcriptase). 2. Insertion into bacterial plasmid to form recombinant plasmid: STICKY ENDS: Same RE used to linearize the vector DNA is used to isolate gene of interest. Ensures sticky ends ARE complementary. DNA ligase then used to anneal. BLUNT ENDS: a) Adding bases: Terminal transferase: single chain of nucleotides can be added to each ends.

b) Linkers DNA: short DNA fragments with known RE sites are cloned to DNA fragment to be cloned to plasmid vector. 3. Transformation: alteration of a bacterial cells genotype or phenotype by the uptake of foreign DNA. Chemical transformation: Chemical competenceCaCl2 solution at 0 deg. Ca2+: creates pores in the membrane: assists binding of the DNA to the cell membrane and masks the negative charges (hydrophilic cell membrane). At 42 deg heat shock, the DNA is forced into the cells. Electroporation: Application of an electric current: momentary pores created

Screening
1. Antibiotic resistance screening 2. Blue-white screening All the MCS are incorporated into LacZ gene. Presence of antibiotic resistance gene, X-gal, and IPTG in cloning vector. Presence of the LacZ gene in plasmid restores the B-galactosidase activity, which converts X-gal into a blue product. Bacteria with non-recombinant plasmid has an intact LacZ gene, and hence can produce functional b-galactosidase, which turns X-gal blue. (due to insertional inactivation as all MCS are located along the LacZ gene. (Only one antibiotic resistance gene required) 4. Hybridization with radioactive nucleic acid probes Probe: Single stranded nucleic acid RNA/DNA, labeled with fluorescent tag Since we know part of the nucleotide sequence of the gene of interest, a probe complementary to it can be synthesized. by forming hydrogen bonds specifically to a complementary sequence, the desired gene is labeled with a radioactive isotope/fluorescent tag.

DNA Libraries
Genomic library Cleaving of an entire genome with a specific RE, same RE used to digest the vector. DNA fragments and open plasmids are ligated together. Recombinant plasmids are transformed into bacteria Screening via radioactive probe cDNA library mRNA has poly A tail, which is used by reverse transcriptase to make the first DNA strand and a stretch of thymine DNA to be used as DNA primer mRNA is degraded by another enzyme

Second DNA complementary to the first is synthesized by DNA polymerase DSDNA is called a cDNA, which is inserted into a vector Uses and differences Contains all DNA sequences: coding, nonCoding sequences: specific to the

coding for eg. Introns, regulatory sequences, telomeric/centromeres etc Non-functional proteins will be produced, as bacterial systems lacks the ability to carry out post-transcriptional modifications: glycosylation Used to obtain cell type/ regulatory sequences/introns associated with a gene

proteins Functional proteins can be produced: non-coding sequences are already absent, do not have to carry out posttranscriptional modifications Coding sequences in gene, studying specialized functions of a particular cell type, changes in patterns of gene expression during development