Molecular Genetics 202 Lecture Notes PDF

MOLECULAR GENETICS 202  ‫رؤﯾﺔ ﻛﻠﯾﺔ اﻟﺗﻛﻧوﻟوﺟﯾﺎ اﻟﺣﯾوﯾﺔ‬ ‫أن ﺗﻛون ﻛﻠﯾﺔ ﻣﻌﺗﻣدة أﻛﺎدﯾﻣﯾﺎ وراﺋدة ﻓﻲ‬ ‫ﻣﺟﺎﻻت اﻟﺗﻛﻧوﻟوﺟﯾﺎ اﻟﺣﯾوﯾﺔ ﻋﻠﻲ اﻟﻣﺳﺗوي‬ ‫اﻟﻣﺣﻠﻲ واﻷﻗﻠﯾﻣﻲ واﻟدوﻟﻰ ‪.‬‬  ‫ر ﺳ ﺎ ﻟ ﺔ ﻛ ﻠ ﯾ ﺔ ا ﻟ ﺗ ﻛ ﻧ و ﻟو ﺟ ﯾ ﺎ ا ﻟ ﺣ ﯾ و ﯾ ﺔ‬ ‫ﺗﻠﺗزم ﻛﻠﯾﺔ اﻟﺗﻛﻧوﻟوﺟﯾﺎ اﻟﺣﯾوﯾﺔ – ﺟﺎﻣﻌﺔ ﻣﺻر ﻟ...

MOLECULAR GENETICS 202  ‫رؤﯾﺔ ﻛﻠﯾﺔ اﻟﺗﻛﻧوﻟوﺟﯾﺎ اﻟﺣﯾوﯾﺔ‬ ‫أن ﺗﻛون ﻛﻠﯾﺔ ﻣﻌﺗﻣدة أﻛﺎدﯾﻣﯾﺎ وراﺋدة ﻓﻲ‬ ‫ﻣﺟﺎﻻت اﻟﺗﻛﻧوﻟوﺟﯾﺎ اﻟﺣﯾوﯾﺔ ﻋﻠﻲ اﻟﻣﺳﺗوي‬ ‫اﻟﻣﺣﻠﻲ واﻷﻗﻠﯾﻣﻲ واﻟدوﻟﻰ ‪.‬‬  ‫ر ﺳ ﺎ ﻟ ﺔ ﻛ ﻠ ﯾ ﺔ ا ﻟ ﺗ ﻛ ﻧ و ﻟو ﺟ ﯾ ﺎ ا ﻟ ﺣ ﯾ و ﯾ ﺔ‬ ‫ﺗﻠﺗزم ﻛﻠﯾﺔ اﻟﺗﻛﻧوﻟوﺟﯾﺎ اﻟﺣﯾوﯾﺔ – ﺟﺎﻣﻌﺔ ﻣﺻر ﻟﻠﻌﻠوم واﻟﺗﻛﻧوﻟوﺟﯾﺎ‬ ‫ﺑﺗﺧرﯾﺞ ﻣﮭﻧدس ﺗﻛﻧوﻟوﺟﯾﺎ ﺣﯾوﯾﺔ طﺑﻘﺎ ﻟﻠﻣﻌﺎﯾﯾر اﻻﻛﺎدﯾﻣﯾﺔ اﻟﻣﻌﺗﻣدة ﯾﻠﺑﻲ‬ ‫اﺣﺗﯾﺎﺟﺎت ﺳوق اﻟﻌﻣل اﻟﻣﺣﻠﻲ واﻻﻗﻠﯾﻣﻲ ﻓﻲ اﻟﻘطﺎﻋﺎت اﻟطﺑﯾﮫ‬ ‫واﻟﺻﯾدﻻﻧﯾﺔ واﻟزاﻋﯾﺔ واﻟﺑﯾﺋﯾﺔ واﺟراء ﺑﺣوث ﻋﻠﻣﯾﺔ ﻣﺑﺗﻛرة وﺗﻘدﯾم‬ ‫ﺧدﻣﺎت ﻣﺟﺗﻣﻌﯾﺔ واﺳﺗﺷﺎرات ﻋﻠﻣﯾﺔ ﻓﻲ اطﺎر ﻗﯾم ارﺗﻘﺎﺋﯾﺔ ‪.‬‬ DNA Manipulative Enzymes By Dr. Hala Eissa Professor of Molecular Genetics 1. Describe the activities and main applications of the different types of enzyme used in recombinant DNA research 2. Differentiate between the different types of DNA manipulative enzymes CUT & join Shortened Lengthened Modified by the add or remove of specific chemical groups Copied into RNA or into DNA DNA manipulative enzymes can be grouped into four broad classes, depending on the type of reaction that they catalyze Nucleases: are enzymes that cut, shorten, or degrade nucleic acid molecules Ligases: join nucleic acid molecules together Polymerases: make copies of molecules. Modifying enzymes: remove or add chemical groups. Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand Hydrogen bond Nucleotide Phosphodiester bond There are two different kinds of nucleases: Exonuclease Endonuclease Exonuclease: remove nucleotides one at a time from the end of a DNA molecule. Cleavage Cleavage Endonuclease: are able to break internal phosphodiester bonds within a DNA molecule Cleavage Difference between Exonucleases The main distinction between different exonucleases lies in the number of strands that are degraded when a double- stranded molecule is attacked Exonucleases Exonuclease III: degrade just one strand of a Bal131: removes nucleotides from both double-stranded molecule, leaving single- strands of a double-stranded molecule stranded DNA as the product 5` 3` 3` 5` Endonucleases a) S1 nuclease: Only cleaves single strands b) DNaseI: Cuts both single and double-stranded molecules DNase 1 is non-specific in that it attacks DNA at any internal phosphodiester bond; the end result of prolonged DNase 1 action is therefore a mixture of mononucleotides and very short oligonucleotides c) Restriction endonucleases : Special group of enzymes called restriction endonucleases cleave double- stranded DNA only at a limited number of specific recognition sites Endonucleases a) S1 nuclease: only cleaves single strands A nick Endonucleases b) DNaseI: cuts both single and double-stranded molecules DNase 1 is non-specific in that it attacks DNA at any internal phosphodiester bond; the end result of prolonged DNase 1 action is therefore a mixture of mononucleotides and very short oligonucleotides Endonucleases c) Restriction endonucleases : special group of enzymes called restriction endonucleases cleave double- stranded DNA only at a limited number of specific recognition sites In the cell the function of DNA ligase is to repair single-stranded breaks ("discontinuities") that arise in double-stranded DNA molecules during, for example, DNA replication. DNA ligases from most organisms can also join together two individual fragments of double-stranded DNA A discontinuity DNA ligase DNA ligase In DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template Primer 5` 3` Newly synthesized strand A T G T A C G T A A C G T A 5` 3` 3` 5` A T G C A T T G C A T T A C G T A A C G T A Template 3` 5`  Four types of DNA polymerase are used routinely in genetic engineering 1. DNA polymerase I This enzyme attaches to a short single-stranded A nick 5` 3` region (or nick) in a mainly double-stranded DNA A T G G C A T molecule, and then synthesizes a completely new T A C G T A A C G T A 3` 5` strand, degrading the existing strand as it proceeds Existing nucleotides are replaced DNA polymerase I is therefore an example of an 5` 3` enzyme with a dual activity - DNA polymerization A T G C A T T G C A T and DNA degradation T A C G T A A C G T A 3` 5`  Four types of DNA polymerase are used routinely in genetic engineering 2.The klenow fragment The polymerase and nuclease activities of DNA polymerase I are controlled by different parts of the enzyme molecule. The nuclease activity is contained in the first 323 amino acids of the polypeptide, so removal of this segment leaves a modified enzyme that retains the polymerase function but is unable to degrade DNA.  Four types of DNA polymerase are used routinely in genetic engineering A nick 2. The klenow fragment 5` 3` A T G G C A T This modified enzyme, called the T A C G T A A C G T A Klenow fragment, can still synthesize a 3` 5` complementary DNA strand on a single- stranded template, but as it has no Existing nucleotides are not nuclease activity it cannot continue the Only the nick is filled replaced in synthesis once the nick is filled in. 5` 3` A T G C A T T G C A T T A C G T A A C G T A 3` 5`  Four types of DNA polymerase are used routinely in genetic engineering 3. Taq DNA polymerase Used in the polymerase chain reaction (PCR) The DNA polymerase I enzyme of the bacterium Thermus aquaticus  Four types of DNA polymerase are used routinely in genetic engineering 4. Reverse transcriptase An enzyme involved in the replication of several kinds of virus 5` 3` A T G Reverse transcriptase is unique in that it uses u a c g u a a c g u a 3` 5` as a template not DNA but RNA RNA Template The ability of this enzyme to synthesize a DNA strand complementary to an RNA New strand of DNA template is central to the technique called 5` 3` A T G C A T T G C A T complementary DNA (cDNA) cloning u a c g u a a c g u a 3` 5` Alkaline phosphatase Polynucleotide kinase Terminal deoxynucleotide transferase  Is a hydrolase enzyme P2-o 3 OH responsible for removing phosphate groups from many HO P 2-o3 types of molecules, including nucleotides, proteins, and alkaloids. HO OH The process of removing the phosphate group is called HO OH dephosphorylation. The most common alkaline phosphatases used in research are: Bacterial alkaline phosphatase (BAP), from Escherichia coli C4 cells Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis) Calf intestine alkaline phosphatase (CIAP), from calf intestine Placental alkaline phosphatase (PLAP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the transmembrane domain) - the secreted alkaline phosphatase (SEAP) Alkaline phosphatase has become a useful tool in molecular biology laboratories : since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step of the process for which they are being prepared; Also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment From E. coli infected with T4 phage Has the reverse effect to alkaline phosphatase, adding phosphate groups onto free 5' termini 2- HO OH o3P OH HO OH HO P 2-o3 From calf thymus tissue Adds one or more deoxyribonucleotides onto the 3' terminus of a DNA molecule Unlike most DNA polymerases it does not require a template. 5` 3` 5` 3` 5` 5` 3` 3` 5` 5` Terminal transferase has applications in molecular biology. It can be used in Rapid Amplification of cDNA Ends (RACE) to add nucleotides which can then be used as a template for a primer in subsequent PCR. It can also be used to add nucleotides labelled with radioactive isotopes. They are able to change the conformation of covalently closed- circular DNA (e.g. plasmid DNA molecules) by introducing or removing supercoils It wraps around DNA and makes a cut permitting the helix to spin. Once DNA is relaxed, topoisomerase reconnects broken strands.