User:Faithmartin/Mutagenesis (molecular biology technique)

(exact text current version)- 2/29/20

Many researchers seek to introduce selected changes to DNA in a precise, site-specific manner. Analogs of nucleotides and other chemicals were first used to generate localized point mutations.[11] Such chemicals include aminopurine, which induces an AT to GC transition,[12] while nitrosoguanidine,[13] bisulfite,[14] and N4-hydroxycytidine may induce a GC to AT transition.[15][16] These techniques allow specific mutations to be engineered into a protein; however, they are not flexible with respect to the kinds of mutants generated, nor are they as specific as later methods of site-directed mutagenesis and therefore have some degree of randomness.

Current techniques for site-specific mutation commonly involve using pre-fabricated mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation or deletion or insertion of small stretches of DNA at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.[17]

The site-directed approach may be done systematically in such techniques as alanine scanning mutagenesis, whereby residues are systematically mutated to alanine in order to identify residues important to the structure or function of a protein.

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Introduction

Site directed mutagenesis allows scientists to alter a specific nucleotide or nucleotide sequence in various animal or human genomes. There are many techniques and outcomes of site directed mutagenesis however two large mechanisms are combinatorial and insertional mutagenesis. Site directed mutagenesis has proved useful in situations that random mutagenesis is not. Random mechanisms such as UV can not target specific regions or sequences of the genome like combinatorial or insertional mutagenesis is capable of. Mutagenesis that is not random has allowed researchers to clone DNA, find the effects of certain mutagens, engineer proteins, help immunocompromised patients, research HIV, fight cancers such as leukemia, and cure beta-thalassemia.

Site Directed Mutagenesis

Random mutagenesis such as UV provide an advantage in terms of control of how many mutations are added. UV mutagenesis allows for the change in single nucleotides, however it does not offer much control as to which nucleotide is being changed. Many researchers seek to introduce selected changes to DNA in a precise, site-specific manner. Prior to site directed mutation, all mutations made were random, following mutation scientists had to use selection for the desired phenotype to attain the desired mutation. However, with new technologies such as cleavage of DNA at specific sites on the chromosome, addition of new nucleotides, and exchanging of base pairs it is now possible to decide where mutations can go[11].The first method used to place mutations on specific locations in DNA was the use of Analogs of nucleotides and other chemicals.[11] Such chemicals include aminopurine. Aminopurine is able to incorporate into DNA in opposite position of the T nucleotide, Aminopurine then causes T to match with C, causing what was originally an A;T base pair, to become a G:C pair therefor being able to exchange specific base pairs.[12] Other molecules such as nitrosoguanidine,[13] bisulfite,[14] and N4-hydroxycytidine may induce a GC to AT transition, giving more options of which mutations can be induced.[15][16] These techniques allow specific mutations to be engineered into a protein; however,they are not as specific as later methods of site-directed mutagenesis and therefore have some degree of randomness.

Current techniques for site-specific mutation commonly involve using pre-fabricated mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation or deletion or insertion of small stretches of DNA at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.[17]

Simplified diagram of the site directed mutagenic technique using pre-fabricated oligonucleotides in a primer extension reaction with DNA polymerase

More current and efficient methods of site directed mutagenesis are being constantly developed. A new technique has been developed called "Seamless ligation cloning extract" (or SLiCE for short) that allows for the cloning of certain sequences of DNA within the genome. This method is now used in Polymerase chain reactions to make site directed mutagenesis more efficient by allowing more than one DNA fragment to be inserted into the genome at once.

The uses for site directed mutagenesis are seemingly endless, however one important function is what the mutations tell us once they have occurred. Site directed mutations were recently used to determine how susceptible certain species were to chemicals that are often used In labs. The experiment used site directed mutagenesis to mimic the expected mutations of the specific chemical. The mutation resulted in a change in specific amino acids and the affects of this mutation were analyzed.

Combinatorial Mutagenesis

(Exact Text Current Version)-03-02-2020

Combinatorial mutagenesis is a technique whereby large number of mutants may be screened for a particular characteristic. In this technique, a few selected positions or a short stretch of DNA may be exhaustively modified to obtain a comprehensive library of mutant proteins. One approach of this technique is to excise a portion of DNA and replaced with a library of sequences containing all possible combinations at the desired mutation sites. The segment may be at an enzyme active site, or sequences that have structural significance or immunogenic property. A segment however may also be inserted randomly into the gene in order to assess the structural or functional significance of particular part of protein.

(Draft of New Text)-03-02-2020

Combinatorial mutagenesis is a site-directed protein engineering technique whereby multiple properties of a protein can be simultaneously engineered based on analysis of the effects of additive individual mutations. It provides a useful method to assess the combinatorial effect of a large number of mutations on protein function. Large numbers of mutants may be screened for a particular characteristic by combinatorial analysis. In this technique, multiple positions or short sequences along a DNA strand may be exhaustively modified to obtain a comprehensive library of mutant proteins. The rate of incidence of beneficial variants can be improved by different methods for constructing mutagenesis libraries. One approach to this technique is to extract and replace a portion of the DNA sequence with a library of sequences containing all possible combinations at the desired mutation site. The content of the inserted segment can include sequences of structural significance, immunogenic property, or enzymatic function. A segment may also be inserted randomly into the gene in order to assess structural or functional significance of a particular part of a protein.

Insertional Mutagenesis

(Exact Text Current Version)-03-02-2020

In cancer research engineered mutations also provide mechanistic insights into the development of the disease. Insertional mutagenesis using transposons, retrovirus such as mouse mammary tumor virus and murine leukemia virus may be used to identify genes involved in carcinogenesis and to understand the biological pathways of specific cancer. Various insertional mutagenesis techniques may also be used to study the function of particular gene.

(Draft of New Text)-03-02-2020

The insertion of one or more base pairs, resulting in DNA mutations, is also known as insertional mutagenesis. Engineered mutations such as these can provide important information in cancer research, such as mechanistic insights into the development of the disease. Retroviruses and transposons are the chief instrumental tools in insertional mutagenesis. Retroviruses, such as the mouse mammory tumor virus and murine leukemia virus, can be used to identify genes involved in carcinogenesis and understand the biological pathways of specific cancers. Transposons, chromosomal segments that can undergo transposition, can be designed and applied to insertional mutagenesis as an instrument for cancer gene discovery. These chromosomal segments allow insertional mutagenesis to be applied to virtually any tissue of choice while also allowing for more comprehensive, unbiased depth in DNA sequencing.

Researchers have found four mechanisms of insertional mutagenesis that can be used on humans. the first mechanism is called enhancer insertion. Enhancers boost transcription of a particular gene by interacting with a promoter of that gene. This particular mechanism was first used to help severely immunocompromised patients I need of bone marrow. Gammaretroviruses carrying enhancers were then inserted into patients. The second mechanism is referred to as promoter insertion. Promoters provide our cells with the specific sequences needed to begin translation. Promoter insertion has helped researchers learn more about the HIV virus. The third mechanism is gene inactivation. A example of gene inactivation is using insertional mutagenesis to insert a retrovirus that disrupts the genome of the T cell in leukemia patients and giving them a specific antigen called CAR allowing the T cells to target cancer cells. The final mechanisms is referred to as mRNA 3' end substitution. Our genes occasionally undergo point mutations causing beta-thalassemia that interrupts red blood cell function. To fix this problem the correct gene sequence for the red blood cells are introduced and a substitution is made.

Overview

- Seamless ligation cloning extract:Used in Polymerase chain reactions to make site directed mutagenesis more efficient by allowing more than one DNA fragment to be inserted into the genome at once.

- Insertional mutagenesis:The insertion of one or more base pairs, resulting in DNA mutations. Used in research regarding cancer and retroviruses. - Combinatorial mutagenesis: A site-directed protein engineering technique used to assess the combinatorial effect of a large number of mutations on protein function.