DNA Amplification Techniques PDF

DNA amplification techniques Medical Biochemistry and Molecular Biology Department Faculty of Medicine – Menoufia University ILOs, intended learning outcomes By the end of this lecture you should be able to: Explain methods of DNA amplification. Point out requirements, steps and uses of P...

DNA amplification techniques Medical Biochemistry and Molecular Biology Department Faculty of Medicine – Menoufia University ILOs, intended learning outcomes By the end of this lecture you should be able to: Explain methods of DNA amplification. Point out requirements, steps and uses of PCR.  DNA amplification techniques  Definition: These are techniques used to give thousands or millions copies of a particular gene.  There are two types: 1. In vivo: i.e. inside living cells, this is known as recombinant DNA technology and molecular cloning. 2. In vitro: i.e. inside a test tube e.g. polymerase chain reaction.  1) Recombinant DNA technology  It is the technology concerned with the recombinant DNA molecules or chimeric molecules.  Chimeric molecule: - Is a DNA molecule containing both human and bacterial DNA.  Requirements for preparation of chimeric molecules: donor DNA, cloning vector, restriction enzymes and ligase enzymes. A. Donor DNA  This is the DNA segment of interest which is chosen and inserted into carrier DNA (vector). B. Cloning vectors  Definition: - It is a DNA molecule to which the target DNA can be recombined and cloned.  Characters of vector: - It must have 2 properties 1. The ability to enter the cell. 2. The ability to replicates.  It includes the following types 1) Plasmids: -  These are small, circular, extra-chromosomal double stranded DNA molecules.  They exist as single or multiple copies within the bacterium.  The natural function of plasmid is to give antibiotic resistance to the host cells.   Can be used to clone DNA segments of less than 10 kb.  Advantages of using plasmids as a cloning vector.  Replicate independent from the bacterial DNA.  The complete DNA sequence of many plasmids is known hence the precise location of restriction enzyme cleavage site.  Plasmids are easily separated from the host chromosomes. 2) Phage (viruses):-  They are type of viruses that live in bacteria.  They have linear DNA molecules into which donor DNA can be inserted at several restriction enzyme sites.   After transferring the target DNA into the viral DNA, the virus will replicate by infecting host cells.  Cell lyses occurs and the multiplied recombined viral DNA will be released.  Can be used to clone DNA segments of about 10-20 kb. 3) Cosmids:-  It is a plasmid containing part of phage virus.  It is used to clone large DNA fragments 35-50 kb. 4) Bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC):- Both will accept and propagates DNA inserts of several hundred kbs.  C. Restriction enzymes  Definition:-These are bacterial enzymes that cut DNA of any source into short pieces in a sequence-specific manner.  Each enzyme recognizes and cleaves a specific double stranded DNA sequence that is 4-7 bp long.  Restriction enzymes can cut both strands at specific sequence called palindrome (formed of symmetrical inverted repeat) producing 2 types of cuts. 1. Blunt ends: - The enzyme breaks the phosphate backbone of the 2 strands at the axis of symmetry. 2. Sticky (staggered) ends: - The enzyme breaks the phosphate backbone of the 2 strands in a way that each strand has an overlapping part.  Technique of DNA cloning  DNA cloning:- Is a method for in vivo amplification of a gene.  A clone: - Is a large population of identical molecules that arise from a common ancestor.  Cloning technique is based on that chimeric or hybrid DNA molecules can be constructed in cloning vectors which then continue to replicate in host cell.  2. The Polymerase Chain Reaction (PCR)  It is sensitive, selective and extremely rapid method of amplifying a target sequence of DNA in a test tube.  Specificity is based on the use of two oligonucleotide primers that hybridize to the complementary sequences on the opposite strands of DNA and flank the target sequence.  Steps: 1. Denaturation (separation): - The DNA sample is first heated to separate the 2 strands. 2. Annealing: - The two primers are allowed to bind, one for each strand, by cooling. 3. Polymerization (extension): - The 2 DNA strands each serve as a template for the synthesis of new DNA from the 2 primers.  Each strand is copied by a Taq polymerase (heat stable DNA polymerase extracted from thermus aquaticus bacteria that can live and replicate at 70 – 80 ᵒC), starting at primer site in presence of dNTPs.  Repeated cycles of heat denaturation, annealing of the primers and extension of the annealed primers with the DNA polymerase result in amplification of DNA segments.  DNA sequences as short as 50–100 bp and as long as 10 kb can be amplified. Twenty cycles provide an amplification of 106 and 30 cycles of 109. Uses of PCR 1. It is used in forensic medicine as PCR allows the DNA in a single cell, hair follicle, or spermatozoon to be amplified and analyzed. 2. Detect infectious agents, especially latent viruses. 3. Make prenatal genetic diagnoses. 4. Detect allelic polymorphisms. 5. Establish precise tissue types for transplants. 6. Study evolution, using DNA from archeological samples evolution, using DNA from archeological samples. 7. For quantitative RNA analysis allow RNA copying and mRNA quantitation by the so-called RT-PCR method (cDNA copies of mRNA generated by a retroviral reverse transcriptase). In vitro amplification of DNA by PCR Reverse transcriptase  It is an RNA-dependent DNA polymerase, capable of synthesizing a DNA strand from an RNA template.  Then, the newly synthesized DNA strand, synthesizes a complementary DNA strand to make a double strand DNA.  The original RNA strand is then degraded by specific RNase H.  This reverse transcriptase enzyme is present in some animal viruses called retroviruses.