Presentation on theme: "Mutation Is a change in the genetic material. Structural change in genomic DNA which can be transmitted from cell to it is daughter cell. Structural."— Presentation transcript:
Mutation Is a change in the genetic material. Structural change in genomic DNA which can be transmitted from cell to it is daughter cell. Structural changes in the genome are subdivided in to: 1.Microscopically detected Those that involve a change to all or part of the chromosome, the change will be visible in the chromosome. 2.Sub-microscopic alteration Those that involve a small segment of genomic DNA, the change involve one or more nucleotide. Types of sub-microscopically detected alterations a)Substitution (Point mutation). b)Deletion / Insertion (Frame shift mutation). c)Dynamic / Unstable.
1.Substitution (Point Mutation) Involve a change in a single nucleotide (single base change) and referred as nucleotide substitution. eg: CGA AGA. In Transition mutation, involve Substitution of a purine to a purine ( A to G OR G to A) or pyrimidine to pyrimidine (In the same group). In Transversion mutation, involve Substitution of purine to pyramidine or visa versa. Types of point mutation: Silent, Missense, Non-Sense.
Types of point mutation a ) Silent: If a mutation of a base pair in a DNA molecule results in another triplet which codes for the same amino acid it is called silent mutation.20-25% of mutations are silent. eg:Valine is encoded by 4 different codons (GUU.GUA,GUC,GUG) any substitution of the third nucleotide will be silent. b) Missense: If a mutation (single bp) in a DNA molecule results in a different amino acid and the synthesis of an altered protein – it's called missense mutation.70-75% cases are missense. The common mutation that cause sickle cell anemia disease involve an A to U misssense transversion. ( Hb A Hb S ) if glutamic acid amino acid changed in to valine amino acid sickle cell anemia.
C) Non-Sense If a mutation in a DNA molecule create a new stop codons (UAA,UAG,UGA), it will leads to termination of protein chain. Shortened chains fail to retain normal activity particularly of stop signal comes before the function domain of the protein. 2-4 % of single base pair changes are non-sense.
2.Frame-Shift Mutation A mutation involves the insertion or deletion of a few nucleotides. a.Involves 3 nucleotides or multiple of 3 No disturbance in the reading frame. b.Involve 1, 2, nucleotides not multiple of 3 disturbance in the reading frame frame shift mutation. In this case, the amino acid sequence subsequent to the mutation shows no resemblance to the normal sequence of the gene. eg: Cystic fibrosis.
3.Dynamic/unstable This consists of increased copy number of triplet repeat sequences and referred as triplet amplification. In sequence,ACG triplet exist normally once, or twice or three times. but when there is mutation,the replication expand more than 100 times, ACG,ACG,ACG….due an error in the DNA replication. First defined in fragile X- syndrome.
The Polymerase Chain Reaction (PCR) What is PCR? 1.It is an in vitro method for amplification of certain DNA or RNA fragment. 2.It is a technique used daily in molecular biology. 3.It provides a good alternative for many currently uesd methods.
What we need for PCR? 1.DNA or RNA as tamplet. 2.Primrs. 3.dNTPs mix ( dATP, dTTP,dCTP, dGTP). 4.Buffer ( with Mg 1.5-2.5 mM) 5.Polymerase enzyme. 6.Thermocycler. For what we use PCR? 1.Detection of mutation. 2.Forensic medicine. 3.Direct cloning of PCR product. 4.Genotyping and diagnosis of infectious diseases HIV, HBV. 5.Mutagenesis by PCR.
Principle of the PCR The purpose of PCR is to enzymaticaly synthesizing and amplifying defined DNA sequences ( make a huge number of copies of a gene) A PCR reaction include the target DNA, a thermostable DNA polymerase, 2 oligonucleotide primers, deoxynucleotide triphosphates (dNTPs),reaction buffer and magnesium. The component are mixed the reaction is placed in the thermal cycler. The thermal cycler takes the reaction through a serious of different temperatures for varying a mounts of time. This serious of temperatures and time adjustments is referred to as one cycle of amplification.
The cycling reactions There are 3 major steps in PCR, which are repeated for up to 45 cycles. This is done in automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. 1.Denaturation at (90-95°C) for 15 second to 2 minutes. During denaturation, the double strand helix open to a single stranded DNA (i.e. the two strands of DNA separate from each other and produce the necessary single stranded DNA template for the thermo stable polymerase.
2.Annealing at (40-72°C) for 30 to 60 seconds. the primer will anneal to the single strand of DNA. The 1 st primer is complementary to the beginning of the original strand sequence. The 2 nd primer is complementary to the end of the sequence (One from up and the other from down) The two Oligonucleotides primer: is short sequence of DNA nucleotide (20-35 nucleotides) that is complementary to the sequence on one strand of the DNA double helix at the opposite ends of the origin to be amplified.
3. Extension or elongation (at 72°C) for 1 to 2 minutes The bases (complementary to the template ) are coupled to the primer on the 3’side( the polymerase add dNTPs from 5’ to 3’, reading the template from the 3’to5’side, bases are add complementary to the template) This step complete the cycle and the next cycle begins with the return to 95°C for denaturation. The cycle will be repeated so, we will make more amount of DNA from 2 DNA we get 4, 8, 16, and 32….etc.