PrepTutorEJDIC

radio frequency無線周波数

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/07/03 04:37:58」(JST)

wiki ja

RFLP（Restriction Fragment Length Polymorphism、制限酵素断片長多型）とは、制限酵素によって切断された DNA 断片の長さが、同一種内の個体間で異なる（多型を示す）ことを指し、また転じてそれを検出する手法も意味する。一塩基多型によってこの違いが現れることもある。遺伝病を持つ人と持たない人でこれが異なる場合があり、診断や遺伝病の原因遺伝子の同定に利用される。得られた断片長は、電気泳動によって既知の断片長のDNA（サイズマーカー）と比較して求める。

ゲノムプロジェクトの進行に伴ってPCRに用いるプライマーの設計が容易になったため、現在ではPCRでDNAを増幅し、その反応生成物を制限酵素で切断する手法であるPCR-RFLPが一般的となった。PCR-RFLP以前はヒトを対象とした場合、

生きた細胞の採取
細胞の培養（数日）
比較的高品質なDNAの抽出
DNAの切断
電気泳動
ブロッティング
放射性同位元素等でラベルしたプローブのハイブリダイズ
X線フィルムへの感光・現像

といった手順を踏んだ。一方、PCR-RFLPでは、

比較的粗雑なDNAの採取
PCR（2時間程度）
DNAの切断
電気泳動
紫外線によるDNAの可視化

と、半日程度で可能となった。

断片長の差で対立遺伝子等を識別する方法

以下にサザンブロッティングを利用した原法を模式的に示す。

個体A
               a                     b         c
               |         2 kb        |   1 kb  |
DNA -----------|---------------------|---------|-------
             SfaN I                SfaN I    SfaN I
                     =========
                   プローブと相補的な領域

個体B
               a                     b         c
               |              3 kb             |
DNA -----------|-------------------------------|-------
             SfaN I                          SfaN I
                     =========
                          

      サザンブロッティングの結果   ↓                 
                                        
         5 kb _____                     
         4 kb _____                     
                                        
         3 kb _____            ______            
                                               
                                              
         2 kb _____   ______                   
                                              
                                               
                                              
         1 kb _____                           
                                        
                                        
         サイズ　      個体A    個体B 
         マーカー                   
                                        
上段: ある生物種2個体のゲノムDNAと制限酵素SfaN Iによる断片長。
         個体Bにはbの位置の制限酵素認識部位が存在しない==はプローブが結合する領域。
下段: サザンブロッティングの結果。個体Aでは2 kbの、個体Bでは3 kbの位置にバンドが見える。
（簡略のため一倍体生物を想定している）

wiki en

[Wiki en表示]

In molecular biology, restriction fragment length polymorphism, or RFLP (commonly pronounced “rif-lip”), is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces and (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. RFLP analysis was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing.

Analysis technique

The basic technique for the detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe. An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis.

RFLP analysis may be subdivided into single- (SLP) and multi-locus probe (MLP) paradigms. Usually, the SLP method is preferred over MLP because it is more sensitive, easier to interpret and capable of analyzing mixed-DNA samples.^{[citation needed]} Moreover, data can be generated even when the DNA is degraded (e.g. when it is found in bone remains.)

Schematic for RFLP by cleavage site loss.

Analysis and inheritance of allelic RFLP fragments (NIH).

Schematic for RFLP by VNTR length variation.

Examples

There are two common mechanisms by which the size of a particular restriction fragment can vary. In the first schematic, a small segment of the genome is being detected by a DNA probe (thicker line). In allele "A", the genome is cleaved by a restriction enzyme at three nearby sites (triangles), but only the rightmost fragment will be detected by the probe. In allele "a", restriction site 2 has been lost by a mutation, so the probe now detects the larger fused fragment running from sites 1 to 3. The second diagram shows how this fragment size variation would look on a Southern blot, and how each allele (two per individual) might be inherited in members of a family.

In the third schematic, the probe and restriction enzyme are chosen to detect a region of the genome that includes a variable VNTR segment (boxes). In allele "c" there are five repeats in the VNTR, and the probe detects a longer fragment between the two restriction sites. In allele "d" there are only two repeats in the VNTR, so the probe detects a shorter fragment between the same two restriction sites. Other genetic processes, such as insertions, deletions, translocations, and inversions, can also lead to RFLPs. RFLP uses a much bigger sample of DNA than STR.

Applications

Analysis of RFLP variation in genomes was a vital tool in genome mapping and genetic disease analysis. If researchers were trying to initially determine the chromosomal location of a particular disease gene, they would analyze the DNA of members of a family afflicted by the disease, and look for RFLP alleles that show a similar pattern of inheritance as that of the disease (see Genetic linkage). Once a disease gene was localized, RFLP analysis of other families could reveal who was at risk for the disease, or who was likely to be a carrier of the mutant genes.

RFLP analysis was also the basis for early methods of Genetic fingerprinting, useful in the identification of samples retrieved from crime scenes, in the determination of paternity, and in the characterization of genetic diversity or breeding patterns in animal populations.

Alternatives

The technique for RFLP analysis is, however, slow and cumbersome. It requires a large amount of sample DNA, and the combined process of probe labeling, DNA fragmentation, electrophoresis, blotting, hybridization, washing, and autoradiography could take up to a month to complete. A limited version of the RFLP method that used oligonucleotide probes was reported in 1985.^[1] Fortunately, the results of the Human Genome Project have largely replaced the need for RFLP mapping, and the identification of many single-nucleotide polymorphisms (SNPs) in that project (as well as the direct identification of many disease genes and mutations) has replaced the need for RFLP disease linkage analysis (see SNP genotyping). The analysis of VNTR alleles continues, but is now usually performed by polymerase chain reaction (PCR) methods. For example, the standard protocols for DNA fingerprinting involve PCR analysis of panels of more than a dozen VNTRs.

RFLP is still a technique used in marker assisted selection. Terminal restriction fragment length polymorphism (TRFLP or sometimes T-RFLP) is a molecular biology technique initially developed for characterizing bacterial communities in mixed-species samples. The technique has also been applied to other groups including soil fungi.

TRFLP works by PCR amplification of DNA using primer pairs that have been labeled with fluorescent tags. The PCR products are then digested using RFLP enzymes and the resulting patterns visualized using a DNA sequencer. The results are analyzed either by simply counting and comparing bands or peaks in the TRFLP profile, or by matching bands from one or more TRFLP runs to a database of known species. The technique is similar in some aspects to DGGE or TGGE.

The sequence changes directly involved with an RFLP can also be analyzed more quickly by PCR. Amplification can be directed across the altered restriction site, and the products digested with the restriction enzyme. This method has been called Cleaved Amplified Polymorphic Sequence (CAPS). Alternatively, the amplified segment can be analyzed by Allele specific oligonucleotide (ASO) probes, a process that can often be done by a simple Dot blot.

External links

http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml
http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf

References

^ Saiki, RK; Scharf S; Faloona F; Mullis KB; Erlich HA; Arnheim N (Dec 20, 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–1354. doi:10.1126/science.2999980. PMID 2999980.

UpToDate Contents

全文を閲覧するには購読必要です。 To read the full text you will need to subscribe.

1. DNAによる親子鑑定 parentage testing using dna
2. 薬剤耐性結核の疫学および分子メカニズム epidemiology and molecular mechanisms of drug resistant tuberculosis
3. B型肝炎ウイルス遺伝子型の臨床的意義 clinical significance of hepatitis b virus genotypes
4. リポ蛋白糸球体症 lipoprotein glomerulopathy
5. 透析患者におけるC型肝炎ウイルス感染の診断 diagnosis of hepatitis c virus infection in patients on dialysis

English Journal

CTLA-4 (+49A/G) Polymorphism and Type-1 Diabetes in Turkish Children.

Celmeli F, Türkkahraman D, Ozel D, Akçurin S, Yegin O.SourceAntalya Education and Research Hospital, Department of Pediatric Immunology, Antalya, Turkey. drdoga@hotmail.com.
Journal of clinical research in pediatric endocrinology.J Clin Res Pediatr Endocrinol.2013 Jun 31. doi: 10.4274/Jcrpe.879. [Epub ahead of print]
Objective: To evaluate the contribution of cytotoxic T-lymphocyte antigen-4 (CTLA-4) +49A/G polymorphism to the susceptibility to type-1 diabetes (T1D) in Turkish children. Methods: A case-control study was designed to include 91 Turkish children with T1D and 99 healthy controls. CTLA-4 (+49A/G) gen
PMID 23367498

Analysis of the human intestinal microbiota from 92 volunteers after ingestion of identical meals.

Jin JS, Touyama M, Kibe R, Tanaka Y, Benno Y, Kobayashi T, Shimakawa M, Maruo T, Toda T, Matsuda I, Tagami H, Matsumoto M, Seo G, Chonan O, Benno Y.SourceBenno Laboratory, Innovation Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Beneficial microbes.Benef Microbes.2013 Jun 1;4(2):187-93. doi: 10.3920/BM2012.0045.
The intestinal microbiota composition of 92 volunteers living in Japan was identified following the consumption of 'identical meals' (1,879 kcal/day) for 3 days. When faecal samples were analysed by terminal restriction fragment length polymorphism with several primer-restriction enzyme systems and
PMID 23271065

Association of single nucleotide polymorphisms in vascular endothelial growth factor gene with bladder cancer risk.

Jaiswal PK, Tripathi N, Shukla A, Mittal RD.SourceDepartment of Urology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow, 226014, India.
Medical oncology (Northwood, London, England).Med Oncol.2013 Jun;30(2):509. doi: 10.1007/s12032-013-0509-8. Epub 2013 Feb 21.
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen involved in a number of pathologic processes, including angiogenesis, tumor growth, and metastasis. Polymorphisms of VEGF gene have been associated with susceptibility to several cancers. VEGF gene susceptibilit
PMID 23430447