Lecture Notes
分子诊断 1 和 2
张咸宁（细胞生物学与医学遗传学系）
2014/09
一、概述
A. Definition
Diagnostic Molecular Genetics = the use of nucleic acid reagents and analytes to
diagnose or predict inherited diseases and carrier states
It is one part of the larger field of DNA Diagnostics/Molecular Pathology, which
also includes nucleic acid-based diagnosis of infectious diseases and cancer, and
"DNA fingerprinting" for purposes of identity testing, establishing parentage,
determining twin zygosity, monitoring transplant engraftment, and forensic
investigations.
It is also one of the two arms of Molecular Medicine, the other being gene
therapy.
B. Unique aspects of Diagnostic Molecular Genetics
Compared to other applications of DNA diagnostics, molecular diagnosis of
genetic disease has a number of unique features:
 examination of an individual's personal genotype
 test results have implications for other blood relatives
 specimens from multiple family members may be required to perform the
analysis (especially for linkage testing)
 can be used to predict disease before onset
 can be used to diagnose disease before birth
二、分子诊断的相关技术
A. Identify Changes in DNA sequence
 Southern blot
o Can detect specific genes in the complex mixture of genomic DNA
o Can detect large deletions, insertion or rearrangements
o Can only detect point mutations which alter restriction enzyme
recognition site
o Can detect gene amplification

Polymerase Chain Reaction (PCR)
o Used as the basis for many sophisticated diagnostic and forensic tests
B. Measuring Gene Expression
 RT-PCR

Real time qPCR
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
Northern blot
o Based upon design of Southern
o Can be used to compare levels of mRNA among samples
o Can detect expression of a gene in specific cells or tissues
o Can detect changes in transcript size
o Can detect changes in the level of expression

DNA Microarrays
o Allows analysis of thousands of genes simultaneously
o Based on cDNA or oligonucleotides bound to glass slides
o One important use is the molecular classification of tumors.
三、分子诊断
A. Classification of genetic disease
The type of approach chosen to diagnosis a genetic disease at the molecular level
is usually based on what is currently known about the causative gene. In increasing
order of diagnostic difficulty, the molecular classes of genetic disease are:
 those for which both the gene and causative mutation are known
 those for which the gene is known, but not the mutation
 those for which neither gene nor the mutation is known
 those caused by more than one gene interacting with environmental factors
(polygenic/multifactorial disorders)
Molecular genetic diagnosis is generally concerned with detection of inherited
(germline) as opposed to acquired (somatic) mutations.
B. Types of clinical molecular genetic applications
Molecular genetic tests may be performed for any of a number of quite different
clinical purposes:
 Clinical diagnosis/confirmation
 Carrier screening（杂合子筛查）
 Prenatal diagnosis
 Newborn screening
 Presymptomatic（症状前） diagnosis/predisposition（易感性） screening
C. Sample collection
Since germline mutations are present in every cell of the body, site-specific
biopsy is not required and simple phlebotomy will suffice for most purposes.
Alternatively, the great sensitivity of PCR allows for genetic testing to be performed
on minute samples of any readily accessible tissue or body fluid, including saliva,
urine, etc. For prenatal diagnosis, several sample types are possible, depending on the
clinical situation:
 Amniocentesis（羊膜穿刺术）: 15th to 16th weeks
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 Chorionic villus samples (CVS。绒毛膜吸取术): 10th to 12th weeks
 Cordocentesis (PUBS。脐穿刺): 19th to 21th weeks
 Embryonic blastomeres（卵裂球） for preimplantation diagnosis (PGD。
植入前遗传学诊断): before the fertilization
 Fetal cells circulating in maternal blood
 cell-free fetal DNA（cffDNA。游离胎儿 DNA）circulating in maternal
blood——NIPT（noninvasive prenatal testing。无创性产前诊断）。一般地，cffDNA
在妊娠第 4 周开始经胎盘进入母体血液循环，约占孕妇血浆游离 DNA 的 10%。
cffDNA 片段可代表胎儿的全基因组。胎儿出生之后，cffDNA 迅速在母体内被清
除。
D. Types of mutations and methods of detection (表 1)
表 1 DNA 突变检测技术一览
检测方法
原理
临床应用
DNA 杂交（Southern）
用 限 制 酶 消 化 基 因 组 可检测 插入 、缺失 和 重
DNA→ 琼 脂 糖 凝 胶 电 泳 排；也可用于绘制物理图
分离 DNA 片段→印迹转 谱
移至尼 龙膜 →与标 记 的
DNA 探针杂交
PCR 产物大小的分析
根据 PCR 产物的大小，选 可检测小插入、小缺失、
择 琼 脂 糖 凝 胶 电 泳 或 三核苷酸重复突变
PAGE
DNA 直接测序
确定 DNA 片段的 4 种碱 可检测插入、缺失、点突
基的线性排列顺序
变、重排
DNA 错配裂解
用标记的 DNA 探针与检 可检测小插入、小缺失、
测的 DNA 进行杂交→在 点突变
碱基错配位点裂解 DNA
ASO
合成特异的互补 DNA 单 可检测 已知 的等位 基 因
链寡核 苷酸 探针 → 标 记 突变
的探针 分别 与样本 进 行
杂交
MLPA
探针与 DNA 靶序列特异 可检测 外显 子或整 个 基
杂交→连接 DNA 片段
因的缺失、重复
质谱法
依据被检 DNA 的有义链 可检测小插入、小缺失、
和无义链单链的物理量
点突变
DNA 微列阵杂交
依据被检 DNA 与 DNA 芯 可检测 SNP、CNV、基因
片的杂交信息
表达谱
蛋白截断实验
纯化被 检组 织或细 胞 的 可检测由于移码突变、间
RNA→用包含 T7 启动子 接位点突变、无义突变导
的 5’引物 RT-PCR 生成 致的蛋白产物截短
cDNA→cDNA 翻译为蛋
白 → 蛋 白 产 物 通 过
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第二代测序（NGS）
SDS-PAGE 进行分析
用一套 寡核 苷酸探 针 来
捕获基 因组 上的目 标 序
列→用 通用 引物对 捕 获
到的序列进行 PCR 扩增
→对扩 增产 物进行 高 通
量测序 生物 信息分 析 →
确定致病基因突变
可同时检测点突变、小插
入、小缺失（<20 bp）、大
片段缺失、重复；适合外
显子的 组成 数目在 十 几
个以上的致病基因；适合
多样本量的同步检测
Usually the laboratory technique chosen will depend on the nature of the
mutation(s) being tested for. Mutation mechanisms to be analyzed in genetic disease
include:
 Point mutations - single nucleotide changes, causing missense or nonsense
coding alterations, or RNA splicing defects
 Microdeletions and insertions - of one or a few nucleotides, causing an amino
acid loss or insertion or (if the change is not an even multiple of three nucleotides) a
frameshift mutation
 Deletions - of large portions of a gene
 Trinucleotide repeat expansions - increase in number of a tandem repeat of
three nucleotides, which may be within an exon, an intron, or a promotor region
It is important to keep in mind that many genetic diseases can be caused by more
than one mutation, by more than one mutational mechanism, or even by more than
one gene. This phenomenon is called genetic heterogeneity（遗传异质性）, and it
makes otherwise straightforward molecular genetic screening tests much more
labor-intensive, expensive, and difficult.
Some techniques are better suited for detecting particular gene alterations than
others. In general, PCR is advantageous because of its extreme sensitivity, its ability
to hone in on small segments of a complex gene, its cost savings in labor and reagents,
and its adaptability to batching of large numbers of specimens. It is not reliable for
examining very large regions of DNA, however, an application for which Southern
blot hybridization is still often required.
To detect point mutations and microdeletions/microinsertions, appropriate
techniques include:
 Southern blot or dot blot hybridization with allele-specific oligonucleotide
(ASO) probes
 Southern blot analysis or PCR amplification, subjecting the products to
digestion with a particular restriction endonuclease whose natural cleavage sequence
is either destroyed or created by the mutation in question
 DNA sequencing
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To detect large deletions, use:
 Southern blot
 Fluorescence in situ hybridization (FISH)
 Routine cytogenetics, if deletion is big enough to be visualized by light
microscopy
（注意：FISH 和常规的细胞遗传学检测都是在“临床细胞遗传学室”而非“分
子遗传学室”进行）
To detect trinucleotide repeat expansions（三核苷酸重复突变）, use:
 Sizing by PCR if the expansion is of moderate length
 Sizing by Southern blot if the expansion is of large size (too large to be
amplified reliably by PCR)
To detect many possible mutations in diseases with marked genetic
heterogeneity, use:
 PCR of several gene regions followed by serial hybridization with a panel of
ASO（等位基因特异的寡核苷酸杂交） probes
 Dot blot hybridization with a pooled cocktail of ASO probes for several rare
mutations at once
 PCR followed by reverse dot blot hybridization to an array of ASO probes
bound to a paper strip or other solid support
 Automated DNA sequencing（自动 DNA 测序）
 Mutation scanning techniques
- Denaturing Gradient Gel Electrophoresis (DGGE)
- Single Strand Conformation Polymorphism (SSCP)
- Denaturing High performance Liquid Chromatography (dHPLC。变性高效
液相色谱)
- High Resolution Melting (HRM)
- Protein Truncation Test (PTT。蛋白截短检测)
 Hybridization to a complex oligonucleotide array ("DNA chip")
All of the techniques listed above are direct mutation detection methods（直接
检测突变的方法）, which can only be used for disorders whose gene has been
identified and cloned. Testing for diseases whose genes have not yet been identified
but have at least been mapped to a particular chromosome requires linkage analysis,
the use of polymorphic DNA markers to follow segregation of the mutant
chromosome in a family group（间接检测突变的方法是利用 DNA 多态性标志进行
相关的连锁分析）.
E. Resources
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Gene Tests: www.genetests.org
American College of Medical Genetics: www.acmg.net
National Society of Genetic Counselors: www.nsgc.org
OMIM: http://www.ncbi.nlm.nih.gov/omim
The journals: Molecular Diagnosis, Diagnostic Molecular Pathology, Journal
of Molecular Diagnosis, Genetic Testing and Molecular Biomarkers, Prenatal
Diagnosis, …
IV. 分子诊断结果可能存在的错误
Molecular genetic testing is generally considered to be extremely accurate, but
errors and misinterpretations can and do occur. The most common sources of
false-negative, false-positive, or indeterminate results are:
 Suboptimal DNA quantity and purity（DNA 抽提的量和纯度不够）
 Incomplete restriction endonuclease digestion
 PCR amplicon contamination（PCR 扩增子的污染）
 Specimen mislabelling and clerical errors
 False paternity（亲子关系不对）
 Recombination between the probe hybridization site and the linked disease
gene (for linkage analysis only)
 Incomplete penetrance（不完全外显） or inconsistent genotype-phenotype
correlations compromising clinical predictive value
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