Download SP01_Lec02_DNA sequencing - bio-bio-1

DNA SEQUENCING
Reading the Blueprint of Life
Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001
Genomic DNA
 All living organisms, from bacteria to human
beings, contain DNA in the nuclei of each of
their cells.
 The blueprint of life exist in the DNA.
 DNA consists of four building blocks or
nucleotides (A, C, G and T)
 Extraction of useful information from genome
sequence of all organisms relies on computeraided data handling –i.e. Bioinformatics.
DNA Preparation
 Whole chromosomes are too large to deal
with, so the DNA is broken into manageably-
sized overlapping segments.
 The DNA segments are cloned using bacteria
and formed a library of DNA segments.
 It is then denatured (ie. melted), so that
the two strands split apart.
DNA Amplification
 In order to sequence a piece of DNA, first need to
amplify it. This is sometimes done by a process
called polymerase chain reaction (PCR).
Denatured DNA is added to a mixture for PCR:
- Primer,
- DNA polymerase
- Nucleotides including special ones called
dideoxynucleotides.
 The dideoxynucleotides are fluorescently tagged.
SEQUENCING USING GEL ELECTROPHORESIS
Here is a gel with 28 DNA samples:
Green:
Blue:
Yellow:
Red:
A
C
G
T
CGACG…………
Small molecules move faster.
Sequence Assembly Using Mapping
 The segments were cut to look for common
sequence landmarks in overlapping fragments.
 These landmarks were used to fingerprint the
fragments so that it was possible to identify the
position of the fragments in the chromosome–
basic strategy of Genome Mapping.
So Genome Mapping is the process of determining
 location of a specific gene in the genome/chromosome
&
 relative distances between genes on the chromosome.
TYPES OF MAPPING
Genetic Mapping: determine the relative
position between two genes on a chromosome.
Physical Mapping: determines the absolute
position of a gene on a chromosome.
Genetic (Genetic Linkage) Mapping
 The genetic linkage map shows the arrangement of
genes and genetic markers along the chromosomes
as calculated by the frequency with which they are
inherited together.
 A genetic map determines a distance between
markers (gene for inherited traits)
 The closer the markers are physically, the more
likely they will be inherited together.
 Markers on genetic linkage map must vary
between individuals.
Commonly Used DNA Markers
 RFLP (Restriction Fragment Length Polymorphism):
RFLP is defined by the presence or absence of a specific site, called a
restriction site, for a bacterial restriction enzyme. This enzyme breaks
apart strands of DNA wherever they contain a certain nucleotide
sequence.
 VNTR (Variable Number of Tandem Repeat Polymorphism):
It occurs in non-coding regions of DNA and is defined by the presence
of a nucleotide sequence that is repeated several times.
Commonly Used DNA Markers
 Microsatellite polymorphism:
It defined by a variable number of repetitions of a very small
number of base pairs within a sequence. The number of repeats
for a given microsatellite may differ between individuals, hence
the term polymorphism (the existence of different forms within
a population).
 SNP (Single Nucleotide Polymorphism):
are individual point mutations, or substitutions of a single
nucleotide, that do not change the overall length of the DNA
sequence in that region. SNPs occur throughout an individual's
genome.
Physical Mapping
 The physical map is representation of the
chromosomes, providing the physical distance
between landmarks on the chromosome, ideally
measured in nucleotide bases.
 Physical maps can be divided into three general
types:
- Chromosomal or cytogenetic maps,
- Radiation hybrid (RH) maps,
- Sequence maps.
The ultimate physical map is the complete
sequence itself.
 Lowest-resolution physical map is the chromosomal or
cytogenetic map, which is based on the distinctive
banding patterns observed by light microscopy of stained
chromosomes.
 Genetic linkage mapping can be used to locate genetic
markers defined by traits observable only in whole
organisms.
 Radiation Hybrid maps are able to provide more
precise information regarding the distance between
markers than can a linkage map.
 The physical map that provides the most detail is the
sequence map.
Cytogenetic Map
Cytogenetic mapping
refers to observing a map
location in reference to a
chromosomal banding
pattern.
Cytogenetic Map
These methods allow a
rough determination of
location, but to not yield a
direct measure of distance.
Radiation Hybrid Mapping
Radiation hybrid
mapping is a method
for high-resolution
mapping.
This data is generally displayed as a vector of numbers or letters…
1 or + for retention
0 or - for non-retention
2 or ? for ambiguous or unknown
Ex.
RN_ALB
RN_HEM
0100110102010001100100100000102210010..
0101110102000100101100200010100110010
Sequence Maps
 95% of genecontaining part of human
sequence finished to 99.99% accuracy
 Sequence maps show genetic markers,
as well as the sequence between the
markers, measured in base pairs.
Common Sources of Sequence Tagged Sites
 Expressed Sequence Tags (ESTs):
short sequences obtained by analysis of complementary
DNA (cDNA) clones.
 Simple Sequence Length Polymorphisms (SSLPs):
arrays of repeat sequences that display length variations.
SSLPs that are polymorphic and have already been
mapped by linkage analysis are particularly valuable
because they provide a connection between genetic and
physical maps.
 Random Genomic Sequences:
they are obtained by sequencing random pieces of cloned
genomic DNA or by examining sequences already
deposited in a database.
References
 http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/A
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pplMapping.shtml
http://www.ncbi.nlm.nih.gov/About/primer/mapping.html
http://www.genome.gov/10000715
http://www.ornl.gov/sci/techresources/Human_Genome/me
dicine/medicine.shtml
http://www.animalgenome.org/edu/gene/index.html
Mapping and Sequencing the Human Genome: Science,
Ethics, and Public Policy. 1992. BSCS and American Medical
Association, Colorado Springs, CO., p. 33.
Bio_Primer_Text_v_0_14_3_1
http://westnilevirus.okstate.edu/research/2004rr/13/13.htm
And ………some downloaded ppt.