Genetic mapping

Gene mapping refers to the process of determining the specific locations of genes on
chromosomes, playing a crucial role in understanding genetic diseases. These maps provide
valuable information about the positions of genes within the genome and the distances between
them, serving as essential tools for genetic research and biotechnology applications. The
history of gene mapping dates back to 1911 when Thomas Hunt Morgan identified the gene
for eye color on the X chromosome of the fruit fly, marking a significant milestone. E.B.
Wilson further contributed by attributing sex-linked genes responsible for color-blindness and
hemophilia in humans to the X-chromosome, aligning with the findings of the Morgan group
in flies.

There are two primary types of gene mapping: genetic mapping and physical mapping. Genetic
mapping relies on genetic techniques to construct maps that illustrate the positions of genes
and other sequence features on a genome, while also helping to determine the relative position
between two genes on a chromosome. Physical mapping, on the other hand, uses molecular
biology techniques to directly examine DNA molecules, with map construction based on these
methods, revealing the positions of sequence features, including genes. A genetic map must
display the positions of distinctive features and requires informative markers that are
polymorphic, along with a population with known relationships, making it most effective when
measured between "close" markers. The unit of distance in genetic maps is centiMorgans (cM),
where 1 cM represents a 1% chance of recombination between markers.

The first genetic map was constructed in the early 20th century, utilizing genes as the initial
markers. The first fruit fly map demonstrated the positions of genes such as body color and eye
color, with all these maps based on the phenotype of the organism. However, visual phenotypes
were limited, and a single phenotype could be influenced by more than one gene, necessitating
more comprehensive and less complex characteristics. For microbes and humans, biochemical
phenotypes were preferred due to the advantage that relevant genes possess multiple alleles, as
seen with the gene HLA-DRB1, which has at least 290 alleles, and HLA-B, which has over
400 alleles.

While genes serve as useful markers, they are not ideal, leading to the development of DNA
markers, which are mapped features that are not genes. DNA markers must have at least two
alleles to be useful, with DNA sequence features satisfying this requirement including
Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Length Polymorphism
(SSLP), and Single Nucleotide Polymorphism (SNP). RFLP, the first type of DNA marker
studied, involves restriction enzymes cutting DNA at specific recognition sequences, though
restriction sites in genomic DNA are polymorphic and exist as two alleles, allowing the RFLP
and its position in the genome map to be worked out by following the inheritance of its alleles.
Scoring an RFLP can be achieved through two methods: Southern hybridization, which
involves transferring DNA fragments to a nylon membrane and hybridizing with a DNA probe
followed by autoradiography to detect specific sequences, and PCR, which uses PCR followed
by restriction digestion and agarose gel electrophoresis to score RFLP utilizing primers to
amplify specific regions.

DNA Markers Methodology for Gene Mapping

Co-dominant DNA markers are a vital methodology in gene mapping, allowing the
simultaneous detection of both alleles at a locus in a heterozygous individual, which is
particularly useful for genetic analysis. This approach relies on markers such as RFLP, Simple
Sequence Length Polymorphism (SSLP), and SNP, which exhibit co-dominance by revealing
the presence of multiple alleles without one masking the other. The process begins with the
extraction of genomic DNA from the organism of interest, followed by the use of specific
techniques to identify variations. For RFLP, restriction enzymes cut the DNA at specific
recognition sequences, and the resulting fragment lengths differ due to polymorphic restriction
sites, which can exist as two alleles. These fragments are then separated using gel
electrophoresis, and the differences are visualized through Southern hybridization or PCR
amplification followed by restriction digestion.

In Southern hybridization, DNA fragments are transferred to a nylon membrane after

electrophoresis, hybridized with a labeled DNA probe, and detected via autoradiography,
enabling the identification of unique band patterns corresponding to each allele. Alternatively,
the PCR method involves amplifying the target DNA region using primers flanking the
polymorphic site, followed by restriction enzyme digestion and agarose gel electrophoresis to
reveal distinct fragment sizes for each allele. SSLP markers, often involving microsatellites,
are analyzed similarly, where the length variations in repeated sequences are amplified by PCR
and separated on gels, with both alleles being detectable in heterozygotes. SNP markers,
detected through techniques like allele-specific PCR or sequencing, also exhibit co-dominance
by identifying single nucleotide differences, with both alleles present in the genotype being
discernible. This co-dominant nature ensures accurate mapping of genetic loci, providing
critical data for constructing genetic maps and studying inheritance patterns, which is essential
for applications in biotechnology such as marker-assisted selection and genetic disease
research.

RFLP is a molecular technique that detects variations in DNA sequences by analyzing

differences in the lengths of restriction enzyme-digested DNA fragments. The principle of
RFLP is based on the fact that restriction enzymes cut DNA at specific recognition sequences,
and polymorphisms in the DNA sequence—such as mutations or insertions/deletions—can
alter the presence or position of these restriction sites. As a result, when DNA from different
individuals is digested by the same restriction enzyme, the resulting fragment lengths can vary.
These fragments are separated by agarose gel electrophoresis, transferred onto a membrane
through Southern blotting, and then hybridized with a labeled DNA probe that targets a specific
genomic region. The resulting band pattern reveals the presence or absence of particular
restriction fragments, which reflect genetic differences among individuals.

In genetic mapping, RFLP serves as a powerful tool to identify polymorphic markers across
the genome. These markers are inherited in a Mendelian fashion, making them useful for
tracking the inheritance of genes and traits. During a typical genetic mapping experiment,
researchers analyze the inheritance pattern of RFLP markers in a population generated by a
controlled cross (such as a test cross). By scoring the presence or absence of RFLP bands in
the progeny and calculating recombination frequencies between markers and phenotypes of
interest, researchers can infer the relative positions of genes on chromosomes. A lower
recombination frequency indicates closer proximity between a marker and a gene, allowing for
the construction of a genetic map expressed in centimorgans (cM).

The primary advantage of RFLP in genetic mapping lies in its high reliability, reproducibility,
and ability to distinguish homozygous from heterozygous individuals due to its codominant
nature. This made RFLP one of the first widely used DNA marker techniques in mapping
studies, especially for identifying disease genes or traits of agricultural importance. However,
RFLP also has limitations: the procedure is labor-intensive, requires large amounts of high-
quality DNA, and involves technically demanding steps such as Southern blotting. With the
advent of faster and more efficient methods like PCR-based markers and SNP genotyping,
RFLP is now less commonly used, though its role in the history of genetic mapping remains
fundamental.

This figure illustrates the principle of RFLP analysis, a molecular technique used to detect
genetic variation. DNA from two samples is first amplified and then treated with a restriction
enzyme. In Sample 1, the enzyme recognizes specific sites and cuts the DNA into fragments,
while in Sample 2, a sequence variation prevents cutting, leaving the DNA intact. When the
digested DNA is run on an agarose gel, Sample 1 shows multiple bands corresponding to
fragments of different sizes, whereas Sample 2 shows a single band representing uncut DNA.
By comparing these banding patterns, RFLP reveals differences in DNA sequences, making it
useful in applications such as genetic fingerprinting, disease diagnosis, and studying genetic
diversity. It can be used as a molecular marker to locate genes on chromosomes. Sequence
variations between individuals can create or abolish restriction enzyme recognition sites,
leading to differences in fragment lengths after digestion. By analyzing these fragment patterns
through agarose gel electrophoresis, researchers can track the inheritance of specific RFLP
markers within families or populations. Since RFLPs are stably inherited, they serve as reliable
landmarks on the genome, allowing the construction of detailed genetic linkage maps and
helping to identify the approximate chromosomal location of genes associated with particular
traits or diseases.
This image illustrates how RFLP is applied in genetic mapping and disease gene
identification. In normal DNA, multiple restriction enzyme recognition sites are present, so
digestion produces several smaller fragments. However, in disease-associated DNA, a
mutation destroys one restriction site, leading to fewer cuts and larger fragments. When these
digested fragments are separated by gel electrophoresis, the normal and disease samples
display distinct banding patterns. A Southern blot with a labeled probe further highlights these
differences, showing specific fragment sizes that distinguish the two DNA types. In genetic
mapping, such RFLP markers act as inherited landmarks across the genome. By analyzing how
these markers segregate with disease traits in families, researchers can identify chromosomal
regions linked to genetic disorders, making RFLPs powerful tools for locating disease-
associated genes.

SSR markers, also known as Simple Sequence Repeats or microsatellites, are short tandem
repeats of 1–6 base pair DNA sequences (e.g., (CA)ₙ, (ATG)ₙ) that are widely distributed
throughout the genome. These regions are highly polymorphic due to variations in the number
of repeat units among individuals, making them extremely useful as genetic markers. The
detection of SSR polymorphisms typically involves designing primers flanking the repeat
region, followed by PCR amplification and gel electrophoresis to separate the resulting
fragments by size. Because the number of repeats varies, individuals will display different
fragment lengths when amplified with the same primer pair.

In genetic mapping, SSR markers serve as highly informative molecular markers due to their
codominant inheritance pattern, which allows for the distinction between homozygous and
heterozygous genotypes. During mapping studies, researchers analyze the segregation of SSR
markers across a population derived from a controlled cross. By scoring the presence or
absence of specific SSR alleles in the progeny and calculating the recombination frequencies
between markers and target traits, scientists can infer the linear order and relative distances of
genes and markers along the chromosome. A low recombination frequency between a marker
and a gene suggests close physical proximity, while a higher recombination frequency indicates
greater distance.

SSR markers offer several advantages in genetic mapping. They are highly polymorphic,
abundant throughout the genome, reproducible, and relatively easy to analyze using PCR,
which requires only small amounts of DNA and is faster than older techniques like RFLP.
Moreover, their codominant nature provides clear genotyping results. As a result, SSR markers
are widely used in plant and animal breeding programs, for gene discovery, and in constructing
high-density genetic maps. However, some limitations exist, such as the need for prior
sequence information to design specific primers and potential challenges in multiplexing many
markers.
The image illustrates the principle of SSR (Simple Sequence Repeat) markers used in genetic
analysis. At the top, three different alleles—Allele A, Allele B, and Allele C—are shown, each
containing a different number of repeat sequences. Allele A has the shortest repeat sequence,
Allele B contains a moderate number of repeats, and Allele C carries the longest repeat
sequence. These differences in repeat number represent natural genetic polymorphisms that
can be detected and used as molecular markers. To analyze these differences, specific primers
are designed to flank the repeat region, and PCR amplification is performed. Because the
number of repeats varies, the resulting PCR products differ in length. These DNA fragments
are then separated by gel electrophoresis based on their size. Smaller fragments, such as those
from Allele A, migrate faster and farther through the gel, while larger fragments, like those
from Allele C, migrate more slowly and stay closer to the well. The image further shows how
genotypes are identified after electrophoresis. For homozygous genotypes, such as AA, BB, or
CC, a single band appears on the gel corresponding to the size of the repeat region for that
allele. In contrast, heterozygous genotypes, such as AC, AB, or BC, display two distinct bands,
reflecting the presence of two different alleles. This clear differentiation allows researchers to
determine the genotype of an individual sample by observing the band pattern.

SNPs are the most common type of genetic variation in the genome, characterized by a single
base-pair change at a specific position in the DNA sequence. For example, one individual may
have a cytosine (C) at a given locus, while another may have a thymine (T). These variations
are typically stable, abundant, and widely distributed across both coding and non-coding
regions of the genome, making them excellent molecular markers for genetic studies.

In genetic mapping, SNPs are used as markers to track the inheritance of genes and traits within
a population. The process begins with genotyping a large number of SNPs across the genome
of individuals from a mapping population, often generated through controlled crosses or natural
populations. Each SNP serves as a distinct landmark. By analyzing the pattern of SNP
inheritance in relation to the trait of interest (such as disease resistance, yield, or physical traits),
researchers can calculate recombination frequencies between SNP markers and the target gene.
A lower recombination frequency indicates that the SNP is physically close to the gene of
interest on the chromosome, while a higher recombination frequency suggests they are farther
apart.

SNP markers offer several advantages over older marker types like RFLP or SSR. They are
highly abundant, can be detected using high-throughput genotyping technologies, and allow
for large-scale automated analysis of hundreds of thousands of loci in a cost-effective and rapid
manner. Additionally, SNPs are typically bi-allelic (having two forms), which simplifies
statistical analysis. This enables the construction of dense genetic maps with high resolution,
facilitating precise localization of genes or quantitative trait loci (QTLs) associated with
important traits.

The four key methods listed are:

The DNA Chip Technology, also called SNP microarrays, is a powerful high-throughput
genotyping method used extensively in genetic mapping and genome-wide association studies
(GWAS). In this approach, thousands to millions of specific oligonucleotide probes
representing known SNP sites are immobilized on a solid glass or silicon surface, forming a
microarray or “DNA chip.” Genomic DNA from the sample is fragmented, labeled with
fluorescent tags, and hybridized to the chip. Only sequences that perfectly match the probes
bind strongly, and a scanner measures the fluorescent signal intensities. Based on these signals,
the presence of specific SNP alleles is determined. DNA chips enable parallel genotyping of a
large number of SNPs in multiple samples simultaneously, making this method highly efficient
for large-scale studies.

The process begins with genomic DNA extraction from biological samples, such as blood,
tissue, or saliva. The extracted DNA contains the complete genetic information of the organism.
Once isolated, the DNA undergoes library preparation, a process where the DNA is
fragmented into smaller, manageable pieces suitable for analysis. This step ensures that the
DNA can effectively hybridize to the probes on the microarray chip. Following library
preparation, the DNA fragments are labeled with fluorescent markers or other detectable tags.
Different samples may be labeled with distinct fluorescent dyes (for example, red and blue),
allowing them to be differentiated later in the analysis. Next, the labeled DNA fragments are
mixed and undergo hybridization by being applied to the microarray chip. The chip contains
thousands of immobilized oligonucleotide probes designed to match specific SNP loci across
the genome. If a DNA fragment matches a probe perfectly (i.e., it contains the specific SNP), it
binds (hybridizes) to that spot on the chip. After hybridization, the microarray chip is scanned
to detect the fluorescent signals from the bound DNA. The intensity and color of each spot
provide information about the presence of particular SNP alleles. These data are visualized as
a microarray result image, where each spot’s color or intensity corresponds to specific SNPs
being present or absent in the sample. Finally, the data are analyzed and interpreted, resulting
in the interpretation of results. This includes plotting SNP genotypes along chromosomes,
detecting patterns of variation, and identifying genetic markers associated with traits or
diseases. The output can be presented graphically, showing SNP distributions and their
relationship to reference genomes.

The Solution Hybridization method involves the hybridization of synthetic, labeled

oligonucleotide probes directly to the target DNA in a solution rather than on a solid substrate.
In this method, complementary probes for each SNP allele are mixed with the sample DNA in
a liquid phase under carefully controlled temperature and salt conditions to allow hybrid
formation. After hybridization, the presence of hybrid complexes is detected by fluorescence
measurement, chemiluminescence, or other signal-detection technologies. This method is
useful when a smaller number of SNPs are analyzed or when flexibility in probe design is
desired. It is often applied when high specificity is required, and rapid genotyping is necessary
without large infrastructure.
Hybridization-based approaches for SNP genotyping. (a) Target hybridization to a probe
array. This method uses allele-specific probes attached to a solid surface for hybridization with
tagged-targets containing SNPs. The surface is washed to remove mismatched targets, then the
genotype of the perfectly matched target-probe pairs is detected by fluorescence. (b) TaqMan®
assay. The assay uses two allele-specific probes with different dyes, reporter (R) and quencher
(Q), at either end with a mismatch at the SNP site. The perfectly matched probe is cleaved
during PCR amplification of the SNP-containing region, release its reporter, and the SNP
genotype indicates by fluorescence analysis

The Oligonucleotide Ligation Assay (OLA) offers a highly specific approach for SNP typing,
leveraging the fact that DNA ligase can only join two adjacent oligonucleotides if they are
perfectly complementary to the target DNA. In this method, two oligonucleotide probes are
designed to hybridize adjacent to each other at the SNP site. One probe is allele-specific and
matches one variant of the SNP perfectly, while the other probe complements the adjacent
region. If the SNP in the sample DNA matches the allele-specific probe, ligation occurs,
producing a joined DNA molecule. The ligated product can be detected via PCR amplification,
fluorescence, or other methods. Because the ligation step is highly selective, OLA provides
very accurate and reliable SNP discrimination, especially useful when distinguishing between
very similar alleles.
Allele specific hybridization. The allele specific hybridization depends on the design of probes.
Allele specific oligonucleotide probes (ASO) have a polymorphic site at the Centre of the probe.
(A) Schematic representation of hybridization of ASO to the target DNA (B) schematic
representation of no hybridization between ASO and target DNA due to mismatch at the
polymorphic site.

The Amplification Refractory Mutation System (ARMS Test), also known as allele-specific
PCR, is a simple yet sensitive SNP genotyping technique. It uses two sets of primers designed
specifically for each SNP allele. Each primer has its 3′-end nucleotide perfectly matching either
the wild-type or the mutant SNP base. During PCR amplification, a primer will only extend
and amplify the target DNA if its 3′-end exactly complements the DNA template at the SNP
position. Thus, depending on which primer yields a PCR product, the genotype can be inferred
as homozygous wild-type, homozygous mutant, or heterozygous. ARMS is widely used due to
its simplicity, low cost, and suitability for analyzing a small to moderate number of SNPs
without requiring sophisticated equipment.
In the given diagram, two scenarios are illustrated. In the first scenario, a G-specific inner
primer perfectly matches the target DNA when the G allele is present at the polymorphic site.
The hybridization occurs successfully, enabling amplification by PCR, which is subsequently
detected. However, if the T allele is present, the G-specific probe fails to hybridize due to the
mismatch at the critical position, preventing amplification. Similarly, a T-specific inner primer
hybridizes only when the T allele is present. The resulting PCR products are visualized using
gel electrophoresis, where different band patterns indicate the genotype. For instance, if both
G and T allele-specific amplifications are successful, the sample is heterozygous (G/T). If only
the G-specific primer produces a band, the genotype is homozygous G/G, and similarly, if only
the T-specific primer produces a band, the genotype is homozygous T/T. This method provides
a rapid, cost-effective, and highly specific approach for SNP genotyping, especially useful for
small-scale studies or focused SNP detection in clinical diagnostics. However, its accuracy
depends on stringent hybridization conditions and precise probe design to minimize cross-
hybridization and false positives.

Physical Mapping

Physical mapping involves the direct examination of DNA molecules using molecular biology
techniques to determine the precise physical locations of genes and other sequence features on
a chromosome. One key technique is the construction of restriction maps, where restriction
enzymes cut DNA at specific recognition sites, and the resulting fragment lengths are analyzed
to establish the order and distance between these sites. This method often utilizes Restriction
Fragment Length Polymorphism (RFLP), where polymorphic restriction sites lead to varying
fragment sizes, which are detected through gel electrophoresis, Southern hybridization, or
PCR-based methods, enabling the mapping of DNA regions based on physical differences.

Southern hybridization is a widely used technique that involves digesting DNA with restriction
enzymes, separating the fragments by gel electrophoresis, and transferring them to a nylon
membrane. A labeled DNA probe is then hybridized to the membrane to detect specific
sequences, and autoradiography visualizes the band patterns, indicating the physical positions
of the target DNA regions. This approach provides a detailed structural view of the genome.
Fluorescence in situ hybridization (FISH) is another method that employs fluorescently labeled
DNA probes to bind to specific chromosomal locations, allowing visualization under a
microscope to map large genomic regions and identify chromosomal abnormalities with high
precision.

Contig mapping is a technique that involves assembling overlapping DNA fragments into a
continuous sequence, often achieved through chromosome walking or the use of bacterial
artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). This process
includes sequencing and aligning the fragments to create a high-resolution map of the genome,
offering a comprehensive view of its physical layout. Pulsed-field gel electrophoresis (PFGE)
is a method that separates large DNA molecules by applying an alternating electric field,
facilitating the mapping of extensive genomic regions with enhanced accuracy by resolving
large fragments that conventional electrophoresis cannot handle. Together, these techniques
form a robust framework for physical mapping, supporting genome analysis and applications
in biotechnology such as gene cloning and genetic disease research.

Fluorescence In Situ Hybridization, commonly known as FISH, is a powerful molecular

cytogenetic technique used for physical mapping of genes on chromosomes. The primary
purpose of FISH in physical mapping is to determine the precise location of specific DNA
sequences or genes on metaphase or interphase chromosomes using fluorescently labeled DNA
probes. In FISH, a DNA probe that is complementary to the target sequence of interest is
labeled with a fluorescent dye. The process begins with the preparation of chromosome spreads
from cells (usually metaphase chromosomes are preferred for high resolution). These
chromosome spreads are then fixed onto a microscope slide, denatured (separating the DNA
strands), and hybridized with the fluorescently labeled DNA probe under specific conditions.
The probe binds (hybridizes) to its complementary DNA sequence on the chromosome. After
hybridization, unbound probes are washed off, and the chromosomes are counterstained (often
with DAPI, which stains all DNA) to visualize the overall chromosomal structure. The slide is
then examined under a fluorescence microscope. The fluorescent signal from the probe
indicates the exact physical location of the target gene or sequence on the chromosome. FISH
provides several advantages for physical mapping. It allows for the direct visualization of gene
location, detection of chromosomal abnormalities (like deletions, duplications, translocations,
or aneuploidies), and is useful even when the genome sequence is incomplete. FISH has been
extensively applied in gene mapping, cancer diagnostics, prenatal testing, and evolutionary
studies. However, the resolution of FISH depends on the size of the probe and the type of
chromosome preparation used. Typically, FISH provides mapping resolution in the range of 1–
10 megabases, although more advanced versions (like fiber-FISH) can offer higher resolution.
FISH is a powerful technique used for physical mapping of genes on chromosomes. The process
begins with the extraction of genomic DNA from cells, which is then fragmented to prepare a
DNA library suitable for analysis. The DNA fragments are labeled with fluorescent dyes, such
as red and blue fluorophores, to enable visualization. These labeled DNA probes are then
hybridized to metaphase chromosome spreads on a microscope slide, where they specifically
bind to their complementary DNA sequences. Alternatively, the labeled probes can be
hybridized to a microarray chip containing thousands of immobilized DNA sequences,
allowing large-scale analysis of multiple genomic regions simultaneously. After hybridization,
the results are visualized as colored spots on the microarray, indicating the presence, absence,
duplication, or deletion of specific genomic regions. The color intensity and pattern of these
spots help in interpreting the physical location of genes or markers, facilitating the
construction of detailed physical maps of the genome. This method is highly useful for detecting
chromosomal abnormalities and understanding gene locations within the genome.

An Expressed Sequence Tag (EST) is a short DNA sequence derived from a complementary
DNA (cDNA) clone that represents part of an expressed gene. Typically ranging from 200 to
800 base pairs, ESTs are generated by sequencing the 5’ or 3’ ends of cDNA, which is
synthesized from messenger RNA (mRNA) isolated from cells or tissues. Because ESTs
originate from mRNA, they correspond to transcribed regions of the genome, providing a
valuable snapshot of gene expression in specific tissues or at particular developmental stages.
In the context of gene mapping, ESTs play a critical role as molecular markers that help
identify the physical location of genes on chromosomes. Researchers can align EST sequences
to a reference genome to determine their precise chromosomal positions. This process is
especially useful for annotating genes, as ESTs often represent exons of protein-coding genes.
Furthermore, by constructing EST libraries from different tissues, scientists can map patterns
of gene expression and investigate how gene activity correlates with phenotypic traits or
diseases. ESTs also assist in positional cloning by narrowing down candidate genes located
within a genomic region linked to a particular trait or disease. Before whole-genome
sequencing became widespread, EST sequencing enabled high-throughput discovery of
expressed genes and accelerated the understanding of gene function and organization. Overall,
ESTs serve as essential tools in gene mapping by bridging the gap between gene expression
data and chromosomal localization, ultimately contributing to gene annotation, comparative
genomics, and the identification of disease-associated genes.

An STS (Sequence Tagged Site) is a short, unique DNA sequence (usually 200–500 base
pairs) that occurs only once in the genome and whose precise location is known. It serves as a
molecular landmark in the genome and is easily detectable by polymerase chain reaction (PCR)
using specific primers. The uniqueness of an STS allows it to be used as a reliable marker in
gene mapping, where the goal is to determine the physical location of genes on chromosomes.
In gene mapping, STSs are extremely useful because they provide reference points across the
genome. By using PCR amplification of an STS from genomic DNA, researchers can quickly
test for the presence or absence of a particular chromosomal region in different individuals or
mapping populations. This makes STSs ideal for constructing physical maps of chromosomes,
especially in large-scale mapping projects such as the Human Genome Project. When many
STSs are mapped and their positions are known, they act like fixed landmarks, enabling
researchers to pinpoint the locations of genes or other genetic elements relative to these
markers. During mapping experiments, geneticists often use a panel of STSs distributed
throughout the genome. By analyzing recombination frequencies in families or mapping
populations, or by aligning STSs to sequence contigs of a reference genome, they can
determine both genetic distances (in centimorgans) and physical distances (in base pairs).
Additionally, STSs can be used in positional cloning of disease genes. For example, if a
particular disease is linked to a chromosomal region, STSs in that region can help narrow down
the candidate interval where the causative gene resides.
STS (Sequence Tagged Site) mapping is a technique used to determine the relative physical
positions of genetic markers along a chromosome. The process begins by generating a
collection of overlapping DNA fragments, which can be derived from techniques such as
restriction enzyme digestion or clone libraries. Each fragment is then analyzed for the presence
of specific STSs using PCR amplification or hybridization methods. In the diagram, two types
of marker pairs are illustrated: a pair of closely linked markers and a pair of less closely linked
markers. For the closely linked markers, the same fragments frequently contain both STSs—
six shared fragments in this example—indicating that these markers are located close together
on the chromosome. In contrast, the pair of less closely linked markers is found together in
fewer fragments—only two shared fragments—suggesting they are farther apart, possibly
separated by large distances or structural elements like the centromere. By assessing how often
pairs of STSs co-occur in the same fragments, researchers can estimate the physical distance
between them. This information allows the construction of a detailed physical map of the
chromosome, which is critical for gene localization, positional cloning, and understanding
chromosome organization. Ultimately, STS mapping provides a powerful approach to link
DNA sequence landmarks to specific chromosomal regions, facilitating gene discovery and
genetic analysis.

Functional Mapping

Functional mapping is a strategy used in genetics to identify the specific locations of genes
on chromosomes that are responsible for particular biological functions or phenotypic traits.
Unlike structural mapping, which focuses on determining the physical position of genes or
markers along the DNA sequence, functional mapping connects the presence or variation of
genes directly to observable traits or biological activities. The process of functional mapping
typically involves analyzing the relationship between genetic variation and phenotype in a
mapping population. Genetic markers such as STSs, microsatellites, or SNPs (Single
Nucleotide Polymorphisms) are used to track inheritance patterns. Researchers perform
statistical analyses, such as Quantitative Trait Locus (QTL) mapping, to associate particular
genomic regions with specific traits of interest, such as disease susceptibility, yield in crops, or
enzyme activity. Functional mapping integrates gene expression data, biochemical pathways,
and molecular interactions to pinpoint genes whose variation leads to functional changes. For
example, in a plant population showing differences in drought tolerance, functional mapping
can identify genomic regions where specific alleles of a gene influence water retention or root
development. By combining linkage data, expression profiles, and functional assays,
researchers can determine not just where a gene is located but also how it contributes to the
trait. This approach is particularly valuable for complex traits that are controlled by multiple
genes (polygenic), where traditional gene identification methods fall short. Functional mapping
helps dissect the genetic architecture of such traits by revealing which loci have the strongest
functional impact and under what environmental conditions they act.

Transcript mapping is the process of determining the physical location of expressed RNA
sequences (transcripts) on a genome. It involves identifying where in the genome a particular
mRNA or cDNA originates from, which helps to link gene expression data to specific
chromosomal positions. This is essential for understanding gene structure, regulation, and
function. The process of transcript mapping typically begins with the isolation of mRNA from
cells or tissues, which is then reverse-transcribed into complementary DNA (cDNA). The
cDNA can be partially sequenced to generate Expressed Sequence Tags (ESTs) or fully
sequenced to represent complete transcripts. These sequences are then aligned to a reference
genome using bioinformatics tools such as BLAST or genome alignment algorithms. The
precise location where the transcript aligns provides the genomic coordinates of the
corresponding gene. Transcript mapping provides several key insights. First, it helps in gene
annotation by identifying exon-intron boundaries and untranslated regions (UTRs), giving a
more complete picture of gene structure. Second, it reveals which genes are actively expressed
in specific tissues or developmental stages. Third, by mapping transcripts from different
conditions (e.g., healthy vs diseased tissues), researchers can identify differentially expressed
genes that may play a role in disease processes. Furthermore, transcript mapping is essential
for alternative splicing analysis. By comparing transcript sequences to the genome,
researchers can detect variations in splicing patterns, which result in different protein isoforms
from a single gene.

Flowchart illustrating the transcript mapping methodology, including key steps: mRNA
isolation from cells, cDNA synthesis, identification of differentially expressed Cdna sequences,
sequencing of cDNA, alignment of sequence reads, and mapping to the reference genome to
identify transcript locations and expression patterns.

Definition and Purpose of Restriction Mapping Restriction mapping is a molecular

biology technique used to create a physical map of a DNA molecule by identifying the
locations of restriction enzyme cleavage sites. Restriction enzymes, derived from bacteria,
recognize specific DNA sequences (typically 4–8 base pairs) and cut the DNA at these sites,
producing fragments of varying lengths. The primary purpose of restriction mapping is to
determine the relative positions of these sites, providing a structural blueprint of the DNA.
This map is critical for applications such as gene cloning, DNA sequencing, or studying gene
organization. For example, knowing the positions of restriction sites helps researchers select
appropriate enzymes for cutting DNA at desired locations during genetic engineering.
Principles of Restriction Mapping The technique relies on the predictable cleavage patterns
of restriction enzymes. Each enzyme cuts DNA at its specific recognition sequence,
generating fragments whose sizes depend on the distance between these sites. By digesting
DNA with one or more enzymes (single or double digests) and analyzing the resulting
fragment sizes, researchers can infer the order and spacing of restriction sites. The process
often involves comparing fragment sizes from different digests to deduce the relative
positions of sites. For instance, a single digest with EcoRI might produce fragments of 2 kb
and 3 kb, while a double digest with EcoRI and HindIII might yield smaller fragments,
revealing additional cleavage points. The data are used to construct either a linear map (for
linear DNA, like PCR products) or a circular map (for plasmids).

Procedure for Restriction Mapping The process begins with isolating the DNA of interest,
such as a plasmid or genomic DNA. The DNA is then digested with one or more restriction
enzymes, either individually (single digest) or in combination (double digest). Digestion is
performed under controlled conditions, ensuring optimal enzyme activity (e.g., correct buffer,
temperature, and incubation time). The resulting DNA fragments are separated using gel
electrophoresis, where smaller fragments migrate faster through the gel than larger ones. The
gel is stained with a dye (e.g., ethidium bromide) to visualize the fragments under UV light,
and their sizes are estimated by comparing them to a DNA ladder (a standard with known
fragment sizes). The fragment sizes from each digest are recorded for further analysis.

Analyzing Fragment Sizes and Constructing the Map To construct a restriction map,
researchers analyze the fragment sizes obtained from single and double digests. For a single
digest, the sum of fragment sizes equals the total length of the DNA molecule. For example,
if a 5 kb plasmid is digested with EcoRI, yielding fragments of 2 kb and 3 kb, there are likely
two EcoRI sites. Double digests provide additional information by showing how sites for
different enzymes are positioned relative to each other. By comparing fragment sizes across
digests, researchers deduce the order and distance between restriction sites. This process can
be complex for large DNA molecules with many sites, requiring logical deduction or
software tools to map accurately. For circular DNA, such as plasmids, the map must account
for the molecule’s topology, ensuring fragment sizes align with a closed loop.

Applications of Restriction Mapping Restriction mapping has numerous applications in

molecular biology. It is used to characterize unknown DNA, such as plasmids or viral
genomes, by providing a fingerprint of restriction sites. In cloning, restriction maps guide the
selection of enzymes to cut DNA at specific sites for inserting genes into vectors. The
technique also aids in verifying recombinant DNA constructs by confirming the presence of
expected restriction sites. Additionally, restriction mapping is used in genetic engineering to
design DNA constructs, in gene mapping to locate genes relative to restriction sites, and in
forensic science for DNA fingerprinting. Although newer techniques like next-generation
sequencing have reduced its prominence, restriction mapping remains a cost-effective and
reliable method for small-scale DNA analysis.

Considerations and Limitations Several factors must be considered when performing

restriction mapping. The accuracy of fragment size estimation depends on high-quality gel
electrophoresis and precise DNA ladders. Incomplete digestion, caused by insufficient
enzyme activity or inhibitors, can produce misleading fragments, so conditions must be
optimized. The presence of multiple restriction sites for the same enzyme can complicate
mapping, requiring additional digests or enzymes with unique sites. For large DNA
molecules, the number of fragments may make manual mapping impractical, necessitating
computational tools. Additionally, restriction mapping assumes that enzymes cut consistently,
but rare events like star activity (non-specific cutting) can skew results. Finally, while
restriction mapping is effective for small DNA molecules like plasmids, it is less practical for
complex genomes, where sequencing is often preferred.

The image illustrates the process of restriction mapping for a 100 kb DNA molecule using
restriction enzymes SalI and BamHI. It shows the DNA being digested with SalI (S), BamHI
(B), and a combination of both (S + B), followed by pulsed-field gel electrophoresis (PFGE)
to separate the resulting fragments. The gel image displays bands for each digest (S, B, S +
B) alongside a DNA marker with sizes ranging from 5 to 100 kb. The fragment sizes observed
are used to construct a restriction map, indicating SalI sites at 60 kb and 10 kb, BamHI sites
at 13 kb, and combined SalI + BamHI sites at 17 kb.

Whole genome shotgun sequencing approach

Whole genome shotgun (WGS) sequencing represents a transformative approach in genomics

that involves determining the complete DNA sequence of an organism's entire genome in a
single, comprehensive process. Unlike targeted sequencing methods that focus on specific
regions, WGS captures every base pair, encompassing both the coding regions (exons) that
directly encode proteins and the vast non-coding regions (introns, regulatory elements,
promoters, and intergenic spaces) that play crucial roles in gene regulation and cellular
function. This method has become increasingly accessible due to advancements in next-
generation sequencing technologies, allowing researchers and clinicians to generate a full
genetic blueprint that can reveal insights into health, disease susceptibility, ancestry, and
evolutionary biology. The process begins with careful sample preparation to ensure high-
quality DNA input, followed by fragmentation, sequencing, and sophisticated bioinformatics
analysis to reconstruct and interpret the genome.

The shotgun sequencing approach begins with the extraction of high-quality DNA from a
biological sample, such as blood, tissue, or environmental material. The DNA is mechanically
or enzymatically sheared into random fragments, typically ranging from 100 to 10,000 base
pairs, depending on the sequencing platform (e.g., short-read platforms like Illumina or long-
read platforms like PacBio). These fragments are then prepared into a sequencing library by
ligating adapters—short synthetic DNA sequences—to their ends, enabling them to bind to the
sequencing platform’s flow cell or nanopore. In some cases, the library is amplified using
polymerase chain reaction (PCR) to increase DNA yield. The fragments are sequenced in
parallel, generating millions to billions of reads, which are short sequences of nucleotides (50–
300 bp for short-read, thousands to millions for long-read). These reads are then
computationally assembled using algorithms, such as overlap-layout-consensus for long reads
or de Bruijn graphs for short reads, to reconstruct the original DNA sequence. If a reference
genome is available, reads are aligned to it using tools like BWA or Minimap2; otherwise, de
novo assembly is performed using software like SPAdes or Canu. Shotgun sequencing can
target entire genomes, as in WGS, or smaller regions, such as plasmids or specific
chromosomes, and is also used in metagenomics to sequence DNA from mixed microbial
communities. The approach is characterized by its random, unbiased fragmentation, which
eliminates the need for prior knowledge of the target sequence.

Advantages of Shotgun Sequencing

One of the primary advantages of shotgun sequencing is its unbiased and comprehensive
coverage, as it randomly fragments DNA, allowing for sequencing without requiring prior
knowledge of the genome or specific primers. This makes it highly versatile, applicable to
whole genomes, individual genes, or complex samples like metagenomes, where the genetic
content is unknown. For example, in microbial genomics, shotgun sequencing can sequence
unculturable bacteria directly from environmental samples, bypassing the need for labor-
intensive culturing. In human genomics, it enables the discovery of novel variants in WGS
projects without targeting predefined regions, unlike gene panels or exome sequencing. This
randomness ensures broad genomic representation, capturing both coding and non-coding
regions when used in WGS, which is critical for studying regulatory elements or structural
variants.

Another key advantage is the high throughput and scalability of shotgun sequencing,
particularly with short-read platforms like Illumina, which can sequence billions of fragments
simultaneously. This allows for rapid data generation, with a single run producing enough reads
to cover a human genome at 30x depth in days. The approach is cost-effective for large-scale
projects, with short-read sequencing costing as low as $600 per human genome in 2025,
making it accessible for population studies like the UK Biobank or microbial diversity projects.
Automation in library preparation and sequencing further enhances scalability, enabling high-
throughput labs to process thousands of samples efficiently, which is ideal for biobanks or
epidemiological studies tracking pathogen genomes during outbreaks.

Shotgun sequencing also offers flexibility across applications, as it can be adapted to various
genomic contexts. In metagenomics, it sequences all DNA in a sample, identifying species,
functional genes, and antibiotic resistance markers without prior selection. In de novo
sequencing of non-model organisms (e.g., rare plants or animals), it reconstructs genomes
without a reference, aiding biodiversity research. For targeted sequencing, it can focus on
specific regions, such as viral genomes or plasmids, making it a universal tool in genomics.
Additionally, the use of long-read shotgun sequencing (e.g., PacBio, Oxford Nanopore)
improves resolution of repetitive regions and structural variants, enhancing assembly accuracy
for complex genomes. This flexibility makes shotgun sequencing a cornerstone of modern
genomics.

The approach benefits from robust computational tools developed over decades, which
streamline assembly and analysis. Algorithms like Velvet, SOAPdenovo, or Canu handle the
complex task of assembling fragmented reads, while alignment tools like Bowtie2 or BWA
map reads to reference genomes with high precision. These tools are supported by extensive
databases (e.g., NCBI, Ensembl) for annotation, making it easier to interpret sequences in
functional or clinical contexts. Moreover, shotgun sequencing data is reusable; once generated,
reads can be re-aligned or re-assembled as new reference genomes or algorithms become
available, providing long-term value for research or diagnostics.

Disadvantages of Shotgun Sequencing

Despite its strengths, shotgun sequencing has notable limitations, particularly in resolving
repetitive or complex genomic regions. Short-read shotgun sequencing (e.g., Illumina)
struggles with repetitive sequences, such as centromeres, telomeres, or tandem repeats, because
short reads (50–300 bp) often cannot span these regions, leading to ambiguous alignments or
assembly gaps. For example, in human genomes, repetitive regions constitute ~50% of the
genome, and misassemblies can result in false negatives for structural variants or incorrect
haplotype phasing. While long-read shotgun sequencing (e.g., PacBio, Oxford Nanopore)
mitigates this by spanning repeats, it introduces higher error rates (up to 5–10% for Nanopore)
and requires additional error-correction steps, increasing computational complexity and cost.

Another significant disadvantage is the high computational demand of shotgun sequencing,

particularly for de novo assembly. Assembling millions or billions of fragmented reads into a
contiguous sequence requires substantial computational resources, including high-performance
servers with large memory and processing power. For example, assembling a human genome
de novo can require hundreds of gigabytes of RAM and days of processing time, even with
optimized algorithms. This complexity escalates for metagenomic samples, where reads from
multiple species must be sorted and assembled, often leading to incomplete or chimeric
assemblies if coverage is uneven. These demands can be a barrier for labs with limited
bioinformatics infrastructure.

Cost, while reduced, remains a limitation, especially for long-read shotgun sequencing or
high-coverage projects. Although short-read sequencing is relatively affordable ($600–$1,000
per human genome in 2025), long-read platforms like PacBio or Oxford Nanopore can cost
$2,000–$5,000 per genome due to lower throughput and expensive reagents. Additionally, the
downstream costs of data storage (100–200 GB per genome) and analysis add to the financial
burden, particularly for large cohort studies or resource-limited settings. For applications like
metagenomics, host DNA contamination (e.g., human DNA in clinical samples) can necessitate
additional enrichment steps, further increasing costs.

Shotgun sequencing also faces challenges with incomplete coverage and sequencing biases.
Random fragmentation can lead to uneven coverage, where some genomic regions are over- or
under-sequenced due to biases in shearing, amplification, or sequencing chemistry (e.g., GC-
rich regions are often underrepresented in Illumina sequencing). Low-coverage shotgun
sequencing (e.g., <10x) risks missing rare variants or producing incomplete assemblies,
limiting its utility for clinical diagnostics. Even at higher coverage, PCR amplification during
library preparation can introduce artifacts, such as duplicate reads, which complicate variant
calling and require additional bioinformatics filtering.

Finally, interpretation challenges arise when shotgun sequencing is used in complex

applications like metagenomics or human genomics. The method generates raw sequence data
without inherent functional context, requiring extensive annotation to identify biologically
relevant variants or species. In metagenomics, distinguishing closely related species or rare
taxa is difficult due to incomplete reference databases, and in human genomics, non-coding
variants identified via shotgun sequencing often have uncertain significance, complicating
clinical utility. These interpretation hurdles demand expertise and access to robust databases,
which may not be available in all settings, limiting the method’s immediate applicability.

The process begins with template DNA, which is randomly fragmented into many small pieces
during the shotgun DNA fragmentation step. Each colored fragment represents a random
piece of the original DNA sequence, generated without a predefined order. Next, the
fragmented DNA undergoes DNA sequencing, where sequencing machines read the nucleotide
sequence of each small DNA fragment. These short reads contain partial DNA information
from random positions in the genome.The next step involves sequence analysis and
reconstruction. The raw sequencing data is analyzed to identify the nucleotide sequence of
each fragment. Two sequencing approaches are illustrated:
  Sanger sequencing, where individual nucleotide signals are read from
chromatograms.
 Next-generation sequencing (NGS), where millions of short reads are produced in
parallel and computationally clustered into overlapping groups.

Finally, the short reads are computationally assembled by aligning overlapping regions,
forming contigs (continuous DNA sequences). These contigs are ordered and merged to
reconstruct the complete assembled genome sequence. The key concept demonstrated in the
image is that shotgun sequencing relies on random fragmentation, high-throughput
sequencing, and computational assembly of overlapping reads to reconstruct an entire genome
without prior knowledge of the sequence. This method provides a rapid and efficient way to
sequence complex genomes, serving as the foundation of modern genome sequencing
technologies.

Clone-by-clone method

The clone-by-clone sequencing method, also referred to as hierarchical shotgun sequencing,

is a systematic approach to sequencing large and complex genomes, such as those of humans
or other eukaryotes. This method was a cornerstone of the Human Genome Project due to its
structured and organized process, which contrasts with the more random approach of whole-
genome shotgun sequencing. It involves breaking the genome into large, manageable
fragments, cloning them into vectors, physical map, and then sequencing and assembling the
fragments. The method ensures high accuracy and is particularly effective for large genomes
with repetitive sequences.

Step 1: DNA Fragmentation The first step in clone-by-clone sequencing involves extracting
high-quality genomic DNA from the target organism and fragmenting it into large pieces.
These fragments typically range from 100 to 300 kilobases (kb) in size, which is large enough
to capture significant genomic regions but small enough to be manipulated in cloning vectors.
Fragmentation is achieved through mechanical methods, such as sonication or nebulization, or
enzymatic methods using restriction enzymes. The goal is to create a collection of overlapping
fragments that collectively represent the entire genome. This step is critical because the quality
and size of the fragments directly influence the efficiency of subsequent cloning and mapping
steps. Care is taken to avoid excessive degradation or shearing to ensure the fragments remain
intact and usable for cloning.

Step 2: Cloning into Vectors Once the DNA is fragmented, the large fragments are inserted
into specialized cloning vectors, such as bacterial artificial chromosomes (BACs) or yeast
artificial chromosomes (YACs). BACs are commonly used due to their stability and ability to
carry inserts up to 300 kb, while YACs can accommodate even larger fragments (up to 1 Mb)
but are less stable. The vectors are introduced into host organisms, typically Escherichia coli
for BACs, where they replicate, producing multiple copies of each DNA fragment. This process
creates a clone library, where each clone contains a unique fragment of the original genome.
The library is stored and maintained for subsequent analysis, ensuring that the genomic
fragments are preserved and can be accessed for mapping and sequencing. This cloning step is
essential for amplifying the DNA and enabling detailed study of each fragment.

Step 3: Physical Mapping Before sequencing, a physical map of the genome is constructed to
determine the relative positions of the cloned fragments. This map serves as a scaffold for
assembling the final sequence. Physical mapping involves identifying overlaps between clones
using techniques such as restriction enzyme mapping, where clones are digested with
restriction enzymes, and the resulting fragment sizes are compared to find overlaps. Another
method is the use of sequence-tagged sites (STSs), which are short, unique DNA sequences
that serve as landmarks to align clones. Techniques like fluorescence in situ hybridization
(FISH) may also be used to anchor clones to specific chromosomal locations. The outcome is
a contig map, a series of overlapping clones (contigs) that collectively cover the genome. This
mapping step is labor-intensive but crucial for ensuring accurate sequence assembly, especially
in genomes with repetitive regions.

Step 4: Subcloning and Shotgun Sequencing Each clone from the library (e.g., a BAC) is
individually processed for sequencing. The large DNA insert in each clone is further
fragmented into smaller pieces, typically 1–2 kb in size, using random shearing or restriction
enzymes. These smaller fragments are cloned into plasmid vectors, creating a subclone library
specific to each BAC. The subclones are then sequenced using Sanger sequencing (historically
the standard method) or, in modern applications, next-generation sequencing technologies.
Sanger sequencing produces reads of 500–800 base pairs, which are highly accurate but limited
in throughput. Each subclone is sequenced multiple times (typically 8–10x coverage) to ensure
reliability. This step generates a collection of sequence reads for each BAC, which represent
the DNA sequence of that particular genomic region.

Step 5: Sequence Assembly The sequence reads from each subclone library are assembled to
reconstruct the sequence of the original BAC clone. This is done using computational tools that
align overlapping reads based on sequence similarity, forming contigs (continuous sequences).
Because the physical map provides the relative positions of the BAC clones, the contigs from
each clone can be aligned to their corresponding genomic locations, facilitating the assembly
of the entire genome. The physical map reduces the complexity of assembly by providing a
framework, which is particularly helpful for genomes with repetitive sequences that can
confuse assembly algorithms. Any gaps or ambiguities in the sequence are resolved through
additional sequencing or targeted PCR amplification. The final output is a complete or near-
complete genomic sequence with high accuracy.

Advantages of Clone-by-Clone Sequencing The clone-by-clone method offers several

advantages, particularly for large and complex genomes. Its reliance on a physical map ensures
high accuracy in sequence assembly, as the map guides the placement of sequenced fragments,
reducing errors in regions with repetitive DNA. The method is also highly reliable for
producing reference-quality genomes, as seen in the Human Genome Project, where it achieved
a high level of completeness and accuracy. By breaking the genome into manageable clones,
the approach simplifies the sequencing process and allows for parallel processing of multiple
clones. Additionally, the clone library serves as a valuable resource for future studies, as clones
can be retrieved and analyzed for specific genomic regions.

Limitations of Clone-by-Clone Sequencing Despite its strengths, clone-by-clone sequencing

has notable limitations. The process is time-consuming and labor-intensive, requiring the
creation of a physical map and the handling of large clone libraries. These steps add significant
cost and complexity compared to whole-genome shotgun sequencing, which skips the mapping
phase. The method also depends on the quality of the clone library and physical map; errors or
gaps in the map can lead to incomplete or inaccurate assemblies. Additionally, some genomic
regions, such as highly repetitive or heterochromatic regions, may be difficult to clone or
sequence accurately. With the advent of high-throughput sequencing technologies, the clone-
by-clone method has become less common for whole-genome sequencing, though it remains
useful for targeted projects or when high accuracy is paramount.

Applications and Historical Context Clone-by-clone sequencing was the primary method
used in the Human Genome Project (1990–2003), which successfully produced the first
complete human genome sequence. Its structured approach was critical for managing the
complexity of the 3-billion-base-pair human genome. The method is still used in specific
contexts, such as sequencing complex regions of genomes, finishing high-quality reference
genomes, or studying specific chromosomal regions. While next-generation sequencing and
long-read technologies have largely replaced clone-by-clone sequencing for whole-genome
projects, its principles remain relevant in genomics, particularly for ensuring accuracy in
challenging genomic regions.

The image illustrates the clone-by-clone sequencing method, a hierarchical approach to

genome sequencing, divided into two panels labeled A and B. Panel A depicts the detailed
process involving multiple cloning steps and mapping, while Panel B shows a simplified
version focusing on a single cloning step. In Panel A, the process begins with the genome,
represented as a horizontal line, which is fragmented into large pieces and cloned into YAC
(Yeast Artificial Chromosome) vectors with an insert size of approximately 1 Mb. These YAC
clones are used to create a low-resolution map. The YAC clones are further subdivided and
cloned into cosmid vectors with inserts of about 40 kb, leading to a high-resolution map.
Finally, the cosmid clones are fragmented into smaller pieces and cloned into plasmid or M13
vectors with insert sizes of 1–10 kb, followed by shotgun sequencing. The resulting sequence
reads are shown as a series of short DNA sequences (e.g.,
...AACTGGCTTATAGCCCTAGCC...). In Panel B, the process is streamlined, starting directly
with the genome, which is cloned into BAC (Bacterial Artificial Chromosome) vectors with
an insert size of approximately 150 kb. These BAC clones are then fragmented and cloned into
plasmid or M13 vectors with insert sizes of 1–10 kb or 1 kb, respectively, and subjected to
shotgun sequencing. The sequence reads are similarly represented as short DNA sequences
(e.g., ...AACTGGCTTATAGCCCTAGCC...). The image highlights the hierarchical nature of
the clone-by-clone method, emphasizing the use of progressively smaller clones and mapping
steps in Panel A, while Panel B presents a more direct approach using BACs. Both panels
converge on shotgun sequencing of small fragments to generate the final sequence data.