第三部分基因组和DNA测序原理优秀课件

1977 Sanger
DNA sequencing via chain termination
1986 Hood; Ansorge
automated DNA sequencing with ‘dye primer’
1989 Murray
‘cycle sequencing’ with Taq polymerase
gene
Exon 1 Exon 2 ... ...
...
Exon n
...
...
protein-coding region
Strategies:
whole genome shotgun (Celera)
random shotgun (HUGO)
Random Shotgun: Genomic library production
Celera: Whole Genome Shotgun Sequencing
whole genome: 3,200,000 kb
random reads (0.6-1 kb)
sheared DNA: 1.0-2.0 kb
sequencing templates
Adapted from: Baylor College of Medicine Human Genome Sequencing Center
mapped BAC clone: 100-500 kb
random reads (0.6-1 kb)
sheared DNA: 1.0-2.0 kb
sequencing templates:
Adapted from: Baylor College of Medicine Human Genome Sequencing Center
32P ATCGATCGAT
32P ATCGATCGA
32P
ATCGATCG
32P
ATCGATC
32P
ATCGAT
32P
ATCGA
32P
ATCG
32P
ATC
32P
AT
32P
A
Polyacrylamide Gel Electrophoresis
T A GC
G C T A
But these bands can’t tell us the identity of the terminal nucleotides
Mapping
MARKERS
MARKERS: ➢ sequence tagged sites (STS) ➢ restriction site pattern
➢ known genes
➢ ...
Mapping of BAC/YAC Clones
Random Shotgun: Sequencing (random phase)
第三部分基因组和DNA测序原 理
人类基因组计划
Human Genome Project
The Human Genome
23 chromosomes 3,2 x109 base pairs
(3,200,000,000) < 5% mRNA (30,000 genes) < 3% translated into protein > 90% unknown function
1991 NEN - DuPONT
‘dye terminator’ sequencing
1999 ABI / MD
96well capillary sequencer
How DNA Sequence Is Determined?
DNA fragments having a difference of one nucleotide can be separated on gel electrophoresis
Shotgun Sequencing: Finishing (both)
The final Goal High Accuracy Sequence:
< 1 error/ 10,000 bases
„finished quality“
Adapted from: Baylor College of Medicine Human Genome Sequencing Center
DNA测序原理
Milestones of DNA Sequencing
1953 Watson, Crick, Franklin molecular structure of DNA
1976 Maxam, Gilbert
DNA sequencing via chemical degradation
whole genome: 3,200,000 kb
partially digested DNA: sizing: 100 - 500 kb
Mapping
pieces: 100-500 kb
BAC libraries
Adapted from: Baylor College of Medicine Human Genome Sequencing Center
➢Sanger双脱氧链终止法
(Sanger和Coulson1977)
新技术方法
➢杂交测序法 ➢质谱法 ➢单分子测序法 ➢原子探针显微镜测序法 ➢流式细胞仪测序法 ➢大规模平行实测法、 DNA 芯片法
方法概述：
生成互相独立的若干组带放射性标记的寡核苷酸，每组 寡核苷酸都有固定的起点，但却随机终止于特定的一种 或者多种残基上。
If those band with the same terminal nucleotide can be grouped, then it is possible to read the whole sequence
DNA测序方法：
经典方法
➢Maxam-Gilbert DNA化学降解法
(Maxam 和Gilbert,1977)
Shotgun Sequencing: Assembly (both)
low base quality
Leabharlann Baidu
single stranded
region
(sequence) gap
= Consensus
mis-assembly (inverted)
„draft quality“
Adapted from: Baylor College of Medicine Human Genome Sequencing Center