Introduction to gene mapping and sequencing . Overview of techniques of gene mapping and sequencing

1. GENE MAPPING
AND SEQUENCING
Preetam Palkar
F.Y. M. Pharmacy
AISSMS college of pharma

2. GENOMICS
• Genomics is the branch of molecular biology concerned with
the structure, functions, evolution and mapping of genome
and sequencing.
• The term genomics was coined by Thomas H. Roderick in
1986.

3. GENETIC MARKERS
• A genetic marker is a DNA sequence with a known physical
location on a chromosome.
• They are used to track the inheritance of a nearby gene that
has not yet been identified but whose approxi location is
known.
• Example
Restriction fragment length polymorphism (RFLP)
Simple sequence length polymorphism (SSLP)
Single nucleotide polymorphism (SNP)

4. GENE MAPPING
• Gene mapping means the mapping of genes to specific
locations on chromosomes
• These maps indicate the locus of genes in the genome and
also distance between them.
• It is very important in case of diagnosis of various genetic
diseases.

5. TYPES OF GENE MAPPING
Genetic mapping or linkage mapping
Relative positions between two genes on a chromosome
Physical mapping
Precise location of specific DNA sequencing which are to be
located .

6. LINKAGE MAPPING
• Gives us an idea whether two genes are linked or not
• Recombination frequency is measured
Recombination frequency = recombinant type/ recombinant
type + parental type *100
Example recombinant frequency = 24 /(24+76)*100 = 24%
• Recombinant frequency is directly proportional to distance
between two genes
• This distance is given in terms of centi Morgan (cM)

8. LOD SCORE
• LOD stands for "logarithm of the odds.“
• In genetics, the LOD score is a statistical estimate of whether
two genes, or a gene and a disease gene, are likely to be
located near each other on a chromosome and are therefore
likely to be inherited.
• A LOD score of 3 or higher is generally understood to mean
that two genes are located close to each other on the
chromosome

9. LOD SCORE

10. PHYSICAL MAPPING
• Also called as Cytogenetic mapping
• Overlapping of genes is studied to construct a map of whole
chromosome
• Techniques of physical mapping
Somatic cell hybridisation
Radiation hybridisation
Fluorescent in situ hybridisation (FISH)

11. SOMATIC CELL
HYBRIDISATION

12. 1. Human cell + mouse cells fusion
2. Formation of hybridoma cells having both genes
3. Elimination of either genes
4. Formation of variety of hybrid cell lines.
5. Subculture to obtain more cell line
6. Subjected to southern blotting.

13. RADIATION CELL
HYBRIDISATION

14. 1. Human cell + mouse cells (irradiated )fusion
2. Formation of hybridoma cells having both genes
3. Elimination of either genes
4. Formation of variety of hybrid cell lines.
5. Subculture to obtain more cell line
6. Subjected to southern blotting

16. 1. Make a probe complementary to the known sequence.
When making the probe, label it with a fluorescent marker,
e.g. fluorescein, by incorporating nucleotides that have the
marker attached to them.
2. Put the chromosomes on a microscope slide and denature
them.
3. Denature the probe and add it to the microscope slide,
allowing the probe hybridize to its complementary site.
4. Wash off the excess probe and observe the chromosomes
under a fluorescent microscope. The probe will show as one
or more fluorescent signals in the microscope, depending
on how many sites it can hybridize to.

17. GENE SEQUENCING
• Gene sequencing is a process in which the individual base
nucleotide in an organisms DNA are identified
• It is the process of determining the precise order of nucleotide
within a DNA molecule
• This sequencing was first proposed by Frederick Sanger in
1975 by chain termination or by dideoxy sequencing
• Allan maxam and Walter Gilbert developed the DNA
sequencing method by chemical modification in 1976

18. METHODS OF GENE SEQUENCING
Basic DNA sequencing
Sanger sequencing
Maxam-Gilbert sequencing
Advanced DNA sequencing
Shot gun sequencing
Next generation DNA sequencing
Solid sequencing
Illumina sequencing
Pyro sequencing

19. SANGER SEQUENCING
• A DNA primer is attached by hybridization to the template
strand and deoxynucleosides triphosphates (dNTPPs) are
sequentially added to the primer strand by DNA polymerase.
• The primer is designed for the known sequences at 3’ end of
the template strand.
• The reaction mixture also contains dideoxy nucleoside
triphosphate (ddNTPs) along with usual dNTPs.
• If during replication ddNTPs is incorporated instead of usual
dNTPs in the growing DNA strand then the replication stops
at that nucleotide.

20. • ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so
it cannot make phosphodiester bond with nest nucleotide,
thus terminates the nucleotide chain
• Respective ddNTPs of dNTPs terminates chain at their
respective site. For example ddATP terminates at A site.
Similarly ddCTP, ddGTP and ddTTP terminates at C, G and T
site respectively.
• The reaction mixture from four batches are loaded into four
different well on polyacrylamide gel and electrophoresed

22. MAXAM-GILBERT SEQUENCING
• Denature a double-stranded DNA to single-stranded by increasing
temperature.
• Radioactively label one 5' end of the DNA fragment to be sequenced by
a kinase reaction using gamma-32P.
• Cleave DNA strand at specific positions using chemical reactions. For
example, we can use one of two chemicals followed by piperdine.
Dimethyl sulphate selectively attacks purine (A and G), while hydrazine
selectively attacks pyrimidines (C and T).
• Now in four reaction tubes, we will have several differently sized DNA
strands.
• Fragments are electrophoresed in high-resolution acrylamide gels for
size separation.

24. SHOT GUN SEQUENCING
• The most efficient way to sequence a large piece of DNA involves a
process known as shotgun sequencing.
• For this, the starting DNA is broken up randomly into many smaller
pieces
• . The resulting sequence reads generated from the different pieces are
then analyzed by a computer program, looking for stretches of
sequence from different reads that are identical with one another.
• When identical regions are identified, they are overlapped with one
another, allowing the two sequence reads to be stitched together.
• This computer process is repeated over and over and over again,
eventually yielding the complete sequence of the starting piece of DNA.

26. ILLUMINA SEQUENCING
Illumina dye sequencing is a technique used to determine the series of
base pairs in DNA , also known as DNA sequencing .
This sequencing method is based on reversible dye-terminators that
enable the identification of single nucleotides as they are washed over
DNA strands.
It can also be used for whole-genome and region
sequencing, transcriptome analysis, meta genomics
small RNA discovery, methylation profiling, and genome-wide protein
nucleic acid interaction analysis.

27. • Illumina sequencing technology works in three steps : amplify ,
sequence and analyze.
• DNA is fragmented and adapters are added that acts as reference point
during amplification ,sequencing and analysis.
• Modified DNA is loaded onto flow cell where amplification and
sequencing takes place .
• The flowcell contains nanowells that space out fragments and help with
overcrowding.
• Each nanowell contains oligonucleotides that provide an anchoring point
for adaptors to attach.

28. • Now cluster formation begins .
• Thousands of copies of each fragment of DNA are formed
and it is done by bridge amplification PCR.
• Now, primers and modified nucleotides are washed onto
chip.
• These nucleotides have reversible 3’ fluorescent blocker so
the DNA polymerase can add only one nucleotide at a time.
• A chemical deblocking step is then used to remove the 3’
terminal blocking group .
• The process continues until full DNA molecule is sequenced.

30. ADVANTAGE
• Due to automated nature of illumine dye sequencing it is
possible to sequence multiple strands at once .
• Illumina uses only DNA polymerase as opposed to multiple
exprnsive enzymes required by other sequencing techniques.

31. NEXT GENERATION SEQUENCING
• Ion torrent sequencing
• Sequencing by oligonucleotide ligation and detect(SOLiD)
• Pyrosequencing
• Illumina sequencing
• Nanopore sequencing
• Mass spectrophotometric sequencing
• Direct visualisation of single DNA molecule by atomic force microscopy
(AFM)
• Single nucleotide cutting
• Readout of cellular gene expression
• Use of DNA chips or micro arrays

32. SOME COMMERCIAL SEQUENCERS
• Roche1454FLX pyrosequencer – pyrosequcencing
• Illumina genome analyser- sequencing by synthesis
• Applied bio system SOLiD sequencer- sequencing by ligation
• Helicon heliscope
• Pacific biosciences SMRT – zero ode waveguide

33. Application
Non invasive
prenatal
testing
Disease gene
identification
Human
disease and
health
Metagenomics
De novo
sequencing
Ribosome
profiling

34. REFERENCE
• National human genome research institute
• Nachimuthu Saraswathy, Ponnusamy Ramalingam, in Concepts and Techniques in
Genomics and Proteomics, 2011
• Slatko, B. E., Gardner, A. F., & Ausubel, F. M. (2018). Overview ofnext-generation
sequencing technologies. Current Protocols in Molecular Biology, 122, e59. doi:
10.1002/cpmb.59
• Kelly L. Williams, in Encyclopedia of Bioinformatics and Computational Biology, 2019
• M.F. Seldin, in Encyclopedia of Genetics, 2001