Page 669 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

31 Molecular Diagnostic Techniques

CHAPTER and Applications

KEY TERMS

clonality genom e next-generation sequencing (NGS)

deoxynucleotide hem atopathology nucleotide
deoxyribonucleic acid (DNA) loop-m ediated isotherm al am pli ca- polym erase chain reaction (PCR)

dideoxy m ethod (Sanger m ethod) tion (LAMP) prim ers
exom e minimal residual disease

LEARNING OUTCOMES

■ Discuss the goal and ndings of the Human Genome Project. Compare DNA sequencing by the Sanger and related analysis by melt-

■ Explain an overview of molecular techniques in hematology. ing curve analysis, pyrosequencing, and capillary electrophoresis.

■ List and give examples of the bene ts and applications of molecular ■ Discuss the Southern blot technique including clinical applications.

techniques in hematopathology. ■ Describe the principle, advantages, and disadvantages of FISH.

Summarize the importance and examples of gene rearrangement ■ Describe the advantages and outline the generalized steps in next-

studies. generation sequencing (NGS).

Discuss the importance of molecular techniques to detection of Explain how microarrays are applied to immunologic testing.

minimal residual disease. ■ Correctly answer end of chapter review questions.

■ Describe characteristics of single nucleotide polymorphisms (SNPs).

■ Describe the principle of the polymerase chain reaction (PCR) ampli- NOTE:

cation technique, including strengths and weaknesses. indicates MLT and MLS core content

■ Compare various PCR adaptations. ■
indicates MLT (optional) and MLS advanced content
■ Identify and brie y describe other ampli cation techniques.

THE HUMAN GENOME PROJECT microscopic recognition o genome regions smaller than 2 to

3 million bp, chromosome stretches su cient to accommo-

T e goal o the Human Genome Project was to sequence the date about 50 to 100 genes. In contrast, gene probing proce-

exact order o the base pairs in a segment o deoxyribonucleic dures are capable o discerning di erences as small as 10 to

acid (DNA) in order to establish our genetic database. Genetic 50 bp in ragments o individual cloned genes. T e strength o

variations associated with speci c disease or increased risk cytogenetics is not in characterizing gene structure but in its

o speci c diseases are the target o genome investigations. utility in locating major rearrangements, which can then be

Te International Human Genome Sequencing Consortium characterized at the gene level by methods or DNA analysis.

published the rst dra o the human genome in the jour- It is estimated that about 19,500 genes are present in

nal, Nature, in February 2001. oday, the study o the human human beings. T e initial method o analysis used by sci-

genome is as complete as it can be. Small gaps that are unre- entists working on the Human Genome Project in 2003 was

coverable with any current sequencing method remain, rst-generation DNA sequencing, Sanger sequencing. oday,

amounting to about 1% o the gene-containing portion o second-generation sequencing or next-generation sequencing

the genome, or euchromatin. T e Human Genome Project (NGS) analyzes millions o ragments o DNA in sequenced

goal was to sequence only nuclear euchromatin regions o unison rom a single patient specimen. Sanger sequencing is

the genome, which makes up about 90% o the genome. T e commonly the technology used to con rm nucleotide changes

other regions o the cell, called heterochromatin ound in observed with NGS. Sanger sequencing developed in 1975 is

centromeres and telomeres, were not sequenced under the still the “gold standard” in sequencing because the error rate

project. with NGS is still airly high. T e creation o NGS plat orms

Gene sizes range rom a ew thousand to several hun- has made sequencing accessible to many laboratories and has

dred thousand base pairs. T is resolution limit precludes expanded the clinical use o nucleic acid sequencing.

653