Molecular techniques 2 Flashcards
PCR AMPLIFIES SELECTED REGIONS OF DNA in vitro.. explain how
a. Region of target DNA to be amplified
- Add oligonucleotide primers
- Heat to separate strands (95 C)
- Cool; primers ANNEAL (55 - 65 C)
- hEAT TTO 72 C to allow DNA synthesis.
If know sequence of parts of gene can amplify in a test tube.
Use multiple copies of a pair of short, chemically synthesised
primers– bind eachDNA strand (3’ ends point at each other).
- REPEAT STEPS 2,3 AND 4
After 25 cycles, the target sequences has been amplified about 10^6-fold - Polymerases add nts to primers
- Polymerisation shuttles back and forth forming exponentially growing number of specific dsDNA.
PRODUCING PCR PRODUCTS with Sticky ends…
- Initial steps of PCR (heats to 95 C, then cool to anneal and synthesise DNA)
- Second round of PCR
- Further rounds of PCR
- Digest with EcoRI
- DNA that arise from regular PCR have BLUNT or near blunt ends ( depends on polymerase used - TA cloning). While blunt end vector with ligase, very INEFFICIENT.
- Create PCR products with sticky ends- use designed PCR primers containing REs sites.
- Digestion of final PCR product with ER produces fragment ready to be inserted into a vector.
DNA copies of mRNA can also be synthesised:
- to analyse genes that encode proteins, mRNA is better predictor of polypeptide sequence than genomic sequence.
- Complementary DNA (cDNA) = a DNA version of an mRNA molecule (more stable)
- cDNA made by an enzyme called Reverse transcriptase. Must isolate RNA from a tissue in which the gene is expressed.
- Intron EXon, gene seg.
- Transcription (in cell)
- Introns removed (in cell)
- OligodT primer anneal tp poly A tail
- Reverse transcriptase
- mRNA
- Single-stranded cDNA
- DNA polymerase copies
- cDNA
- 3’ Double-stranded
- 5’ cDNA
NOTE: When mRNA strand has degraded (RNAseH), addition of second primer
(Not shown) premits initiation by DNA polymerase, completes synthesis.
Producing cDNA molecules with sticky ends…
- Double stranded cDNA
- Ligate oligonucleotide
-linkers containing - EcoRI sequence
Cut with EcoRI
Ligate into vector cut with EcoRI
Adding EcoRI sites to end of cDNA molecule.
Linkers or adaptors (boxed) added to both ends of cDNA molecule.
CLONING GENES:
One way to amplify a specific piece of DNA is to place fragment into bacterial cell.
termed “gene cloning” as identical copies (clones) of original DNA produced.
A cloning vector is a stable, replicating DNA molecule foreign DNA can be attached 3 important features:
- Origin of replication (allows DNA replication)
- Selectable markers (e.g. antibiotic resistance)
- Unique restriction enzyme sites into which DNA fragment inserted (MCS = multiple cloning site)
PLASMID VECTORS:
Circular DNA molecules exist naturally in bacteria
Origin of replication, so can replicate independently of bacterial chromosome.
Ones used in cloning specially constructed to contain multiple RE sites.
What should plasmid vector s have?
- Origin of replication recognised in the host cell so that replicated along with the DNA that it carries.
- Should carry selectable markers - traits that enable cells containing the vector to be selected or identified.
- Single cleavage site for each of 1 or more restriction enzyme used.
Method for inserting foreign DNA into plasmid vector:
TO CREATE RECOMBINANT DNA
- Both DONOR AND VECTOR DNAs digested by a RE that produces complementary sticky ends
- Resulting fragments are mixed in test tube to allow sticky ends to hybridise.
Amplification of donor DNA inside a Bacterial cell:
A single recombinant vector enters bacterial cells and is amplified by replication machinery -> MANY COPIES OF EACH VECTOR IN BACTERIA
After amplification, a colony of bacteria will contain billions of copies of donor DNA insert fused to vector (CLONE)
AMPLIFICATION STEPS:
- choose cloning vector and insert DNA of interest
- introduce recombinant DNA to bacteria
- Recover amplified recombinant molecule.
CHOICE OF CLONING VECTOR - numerous vectors suitable for different size inserts
Plasmid - Size of DNA that can be cloned: As large 15 kb. Method propagation: Plasmid replication, INTRODUCTION TO BACTERIA: TRANSFORMATION.
Phage lambda - as large as 23 kb, Phage reproduction, phage infection.
Cosmid, 44 kb, plasmid reproduction, phage infection.
Bacterial artificial chromosome..300kb, plasmid reproduction, electrporation.
PLASMID VECTORS
- Small, circular DS DNA replicate INDEPENDENTLY of bacterial chromosome.
- Plasmid vectors usually contain a gene for drug resistance (eg ampR) and a Gene to distinguish plasmid with and without insert ( eg lacZ)
Multiple cloning site.
Understanding Bacteriophage Vectors
;
- Harbours DNA as insert “packaged” inside phage particle.
In lambda - the nonessential central region of phage chromosome discarded
Ends ligated to random 15kb (can be up to 23kb) fragments of donor DNA.
Linear multimer forms, then stuffed into phage heads as monomer.
FOSMIDS (type of cosmid vector) and BACs are cloning vectors that carry large inserts
- Vectors that carry 35kb to 45kb inserts
- engineered hybrids of lambda phage DNA and bacterial plasmid.
- packaged into Lambda phage, which act as syringe and introduce DNA into bacteria
- when in cell, these hybrids form circular molecules, replicate extrachromosomally (like plasmids)
- very few copies accumulate in cell
BACs: Bacterial artificial chromosomes = 3
- most popular cloning vector for large DNA inserts
- Derived from F plasmid (controls mating/transfer genetics material), cam carry 100-200kb inserts, vector itself only 7kb.
- Large circular recombinant DNA introduced into bacteria.
