Protocols in Human Molecular GeneticsExtraordinary advances have been made in the field of human molecular genetics during the past five years. The ability to amplify a specific region ofDNA a millionfold in a few hours using the polymerase chain reaction has led to the rapid identification of mutations in human disease and of DNA sequence polymorphisms on every human chro- some. DNA fragments of up to 1 megabase in length can now be resolved by pulsed-field gel electrophoresis to create long-range physical maps of important regions of the genome, and can be cloned in the form of yeast artificial chromosomes. The discovery of highly variable "minisatellite" DNA sequences has led to the development of DNA fingerprinting. The application of these techniques to the study of the human genome has culminated in major advances such as the cloning of the cystic fibrosis gene, the construction of genetic linkage maps of each human chro- some, the mapping of many genes responsible for human inherited d- orders, genetic fingerprinting of forensic specimens, and the detection of mutations involved in the development of human tumors. Although many of the new techniques in molecular genetics can be learned relatively easily, it is often difficult for a researcher to obtain all of the relevant information necessary for getting up a technique and applying it successfully. The information available in the research lite- ture often lacks the depth of description that the new user requires. |
From inside the book
Results 1-5 of 69
Page 3
... concentration ( see below and Section 6 ) . The optimal number of DNA molecules in the template is between 105 and ... concentration . Amplify the template with the following concentrations of Mg2 + : 1.5 ( as above ) ; 3.0 ; 4.5 ; 6.0 ...
... concentration ( see below and Section 6 ) . The optimal number of DNA molecules in the template is between 105 and ... concentration . Amplify the template with the following concentrations of Mg2 + : 1.5 ( as above ) ; 3.0 ; 4.5 ; 6.0 ...
Page 4
... concentration , 10 % ) may allow successful amplification , but this is not recommended in other cases , since it decreases the efficiency of the poly- merase enzyme by about 50 % ( 5 ) . Addition of an overlay of inert mineral oil ...
... concentration , 10 % ) may allow successful amplification , but this is not recommended in other cases , since it decreases the efficiency of the poly- merase enzyme by about 50 % ( 5 ) . Addition of an overlay of inert mineral oil ...
Page 7
... Concentration of dNTPs or of enzyme too high Annealing temperature too low for GC content of primers " Overamplification Reamplification of primary amplified product Reduce number of cycles ; reduce exten- sion time See note a Reduce ...
... Concentration of dNTPs or of enzyme too high Annealing temperature too low for GC content of primers " Overamplification Reamplification of primary amplified product Reduce number of cycles ; reduce exten- sion time See note a Reduce ...
Page 15
... concentration of some of the ingredients may need to be carefully optimized to ensure most efficient and specific amplifica- tion . However , the " Cetus buffer " has the advantage that it is more likely to be compatible with subsequent ...
... concentration of some of the ingredients may need to be carefully optimized to ensure most efficient and specific amplifica- tion . However , the " Cetus buffer " has the advantage that it is more likely to be compatible with subsequent ...
Page 18
... concentrations of nucleotides that must be used . We have not encoun- tered a region of DNA secondary structure that could not be resolved by T7 DNA polymerase sequencing at 50 ° C , and believe that the only ad- vantage of Taq will be ...
... concentrations of nucleotides that must be used . We have not encoun- tered a region of DNA secondary structure that could not be resolved by T7 DNA polymerase sequencing at 50 ° C , and believe that the only ad- vantage of Taq will be ...
Contents
9 | |
21 | |
29 | |
39 | |
Rapid Methods for Detection of Polymorphic Markers in Genomic | 51 |
The Analysis of Point Mutations Using Synthetic Oligonucleotide | 69 |
Detection of Mutations by the Amplification Refractory Mutation | 77 |
Automated Gene Detection Using the Oligonucleotide Ligation | 85 |
Yeast ArtificialChromosome YAC Cloning Systems Michele Ramsay | 197 |
Gene Targeting for Somatic Cell Manipulation Julia R Dorin | 223 |
In Situ Hybridization of Chromosomes Kong H Choo Ruth M Brown | 233 |
Methodology and Its Applications | 255 |
DNA Fingerprinting and Forensic Medicine Karen M Sullivan | 273 |
The Detection of Point Mutations in Hemoglobin Defects Using | 287 |
Detection of Gene Deletions Using Multiplex Polymerase | 299 |
Application of PulsedField Gel Electrophoresis to Genetic Diagnosis | 313 |
Detection of Point Mutations by DenaturingGradient | 95 |
The Detection and Mapping of Point Mutations by RNase | 111 |
Discontinuous Polyacrylamide Gel Electrophoresis of | 123 |
Extraction and Enzymatic Amplification of DNA from Paraffin | 133 |
The Use of the Polymerase Chain Reaction in the Mapping | 141 |
An Update Michael R Evans Andrew L Bertera | 147 |
The Detection of Specific DNA Sequences by Enhanced | 159 |
PulsedField Gel Electrophoresis Johan T den Dunnen | 169 |
Cloning from Gels Following PulseField Gel Electrophoresis | 183 |
Molecular Diagnostics of Cancer Bryan D Young | 327 |
The Detection of Latent Virus Infection by Polymerase Chain | 347 |
Mapping Inherited Diseases by Linkage Analysis Martin Farrall | 365 |
Diagnosis of Genetic Disorders with Linked DNA Markers | 389 |
Software for Genetic Linkage Analysis Stephen P Bryant | 403 |
Creating Animal Models of Genetic Diseases Robert P Erickson | 419 |
Ethical Implications | 437 |
Appendix | 457 |
Common terms and phrases
Acad acrylamide agarose gel aliquots allele amplification annealing approx assay bands cDNA cells centrifuge Chapter chromosome cloning concentration containing deletion denaturing detection DGGE diagnosis digestion disease DNA fingerprinting DNA fragments DNA samples DNA sequence Duchenne muscular dystrophy dystrophin EDTA ethanol ethidium bromide filter gel electrophoresis gene genomic DNA human genome Human Molecular Genetics Incubate labeled Lane ligation linkage analysis loci lod score markers membrane mg/mL minisatellite mismatch mixture mouse NaCl Natl Nucleic Acids Res nucleotide oligonucleotide PCR products pellet PFGE pipet plate point mutations polyacrylamide gel polymerase chain reaction polymorphisms prepared probe Proc Protocols in Human recombination region restriction enzyme resuspend RFLPs room temperature screening Southern blotting specific stained stock solution stored Taq polymerase target technique template tion tissue translocation Tris-HCl tube vector wash yeast µg/mL