DNA,

MUTATION

DETECTION

TYPES OF DNA

There are two major types of DNA: Genomic DNA and Mitochondrial DNA

Genomic DNA / Nuclear DNA:

Comprises the genome of an organism and lead to an expression of genetic traits.

Controls expression of the various traits in an organism.

Sequenced as part of the Human Genome Project to study the various functions of the different regions of the genome

Usually, during DNA replication there is a recombination of genes bringing about a change in sequence leading to individual specific characteristics.

This way the difference in sequence could be studied from individual to individual.

MITOCHONDRIAL DNA(MT DNA)

mtDNA is a double stranded circular molecule.

mtDNA is always Maternally inherited.

Each Mitochodrion contains about 2-10 mtDNA molecules.

mtDNA does not change from parent to offspring (Without recombination)

Containing little repetitive DNA, and codes for 37 genes, which include two types of ribosomal RNA, 22 transfer RNAs and 13 protein subunits for some enzymes

HUMAN STRUCTURAL GENE

1. Helix–turn–helix 2. Zinc finger 3. Leucine zipper 4. Helix–loop–helix

GENE TO PROTEIN

Facilitate transport of the mRNA to the cytoplasm and attachment to the ribosome

Protect the mRNA from from 5' exonuclease Acts as a buffer to the 3' exonuclease in

order to increase the half life of mRNA.

TRANSLATION

TYPES OF DNA SEQUENCE VARIATION

VNTR: Variable Number of Tandem Repeats

or minisatellite (Telomeric DNA, Hypervariable minisatellite DNA)

~6-100 bp core unit

SSR : Simple Sequence Repeat

or STR (short tandem repeat)

or microsatellite

~1-5 bp core unit

SNP: Single Nucleotide Polymorphism

Commonly used to also include rare variants (SNVs)

Insertions or deletions

INDEL – small (few nucleotides) insertion or deletion

Rearrangement (inversion, duplication, complex rearrangement)

CNV: Copy Number Variation

SNP

Most are “silent”

Intragenic

Promoters and other regulatory sequences

Introns

Exons

5’ and 3’ untranslated regions

Coding sequence (~1-2% of genome)

Allele 1 A U G A A G U U U G G C G C A U U G A A C

Allele 2 A U G A A G U U U G G T G C A U U G A A C

A

G

COPY NUMBER VARIATION (CNV)

Kb to Mb in size (average ~250 Kb)

>>2000 known, affect ~12% of human genome

? ~100 / person

Role in human disease/normal traits

MUTATIONS

SILENT SEQUENCE CHANGE (SYNONYMOUS SNP)

Normal mRNA Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

Sequence variant

mRNA Protein

A U G

Met

A A G

Lys

U U U

Phe

G G U

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

G

Changes that do not alter the encoded amino acid

Missense

Missense: changes to a codon for another amino

acid (can be harmful mutation or neutral variant)

mRNA Protein

Normal mRNA Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A U G

Met

A A G

Lys

U U U

Phe

A G C

Ser

G C A

Ala

U U G

Leu

A A

Gln

C

A A

Gln

C

Missense Mutation (Nonynonymous SNP)

Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein

Nonsense

mRNA Protein

Normal mRNA Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A U G

Met

U A G U U U G G C G C A U U G

A A

Gln

C

A A C

Nonsense Mutation (Nonynonymous SNP)

Frameshift U G C A A A U G

Met

A A G

Lys

G C G

Ala

C A U U U G

Leu

Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually)

shortened protein

mRNA Protein

Normal mRNA Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

Frameshift Mutations

Exon 1 Intron Exon 2 Intron Exon 3

Exon 1 Exon 3 Altered mRNA

Splice-site mutation: a change that results in altered RNA sequence

Exon 2

Splice-site Mutations

CHROMOSOMAL DISORDERS

50% of 1st trimester miscarriages

5% of stillbirths

0.5% of liveborns

Down syndrome—trisomy 21

Fragile X syndrome

Somatic cell abnormalities in cancers

Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis.

There are many types of chromosome anomalies.

They can be organized into two basic groups, numerical and structural anomalies.

NUMERICAL ABNORMALITIES

Loss or gain of one or more chromosomes, referred to as aneuploidy

The addition of one or more complete haploid complements, known as polyploidy

STRUCTURAL ABNORMALITIES

Deletions: A portion of the chromosome is missing or deleted.

Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material

Translocations: A portion of one chromosome is transferred to another chromosome. There are two main types of translocations:

Reciprocal translocation: Segments from two different chromosomes have been exchanged.

Robertsonian translocation: An entire chromosome has attached to another at the centromere - in humans these only occur with chromosomes 13, 14, 15, 21 and 22

STRUCTURAL ABNORMALITIES

Inversions: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted and sequence is disturbed

Paracentric: involves only one arm of the chromosome

Pericentric: segment involves the centromere

Insertions: A portion of one chromosome has been deleted from its normal place and inserted into another chromosome.

Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material.

Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.

STRUCTURAL ABNORMALITIES

RECIPROCAL TRANSLOCATION

ROBERTSONIAN TRANSLOCATION

CHROMOSOME NOMENCLATURE

MOSAICISM & CHIMERISM

Mosaicism:The presence in an individual, or in a tissue, of two or more cell lines that differ in their genetic constitution but are derived from a single zygote

Chimerism:The presence in an individual of two or more genetically distinct cell lines derived from more than one zygote

Dispermic Chimeras: two genetically different sperm fertilize two ova and the resulting two zygotes fuse to form one embryo.

Blood Chimeras: result from an exchange of cells, via the placenta, between non-identical twins in utero

METHODS OF CHROMOSOME ANALYSIS

Patient cells are incubated and divide in tissue culture.

Phytohemaglutinin (PHA): stimulates cell division

Colcemid: arrests cells in metaphase

3:1 Methanol:Acetic Acid: fixes metaphase chromosomes for staining

Giemsa-, reverse- or centromere-stained metaphase chromosomes

PREPARATION OF A KARYOTYPE

G-BANDED METAPHASE SPREAD

KARYOTYPE

Probe

Interphase or metaphase cells on slide (in situ)

Microscopic signal (interphase)

FLUORESCENT IN SITU HYBRIDIZATION (FISH)

Hybridization of complementary gene- or region-specific fluorescent probes to chromosomes.

USES OF FLUORESCENT IN SITU HYBRIDIZATION (FISH)

Identification and characterization of numerical and structural chromosome abnormalities.

Detection of microscopically invisible deletions or duplication.

Detection of sub-telomeric aberrations.

Prenatal diagnosis of the common aneuploidies (interphase FISH).

FISH PROBES

Chromosome-specific centromere probes (CEP)

Hybridize to centromere region

Detect aneuploidy in interphase and metaphase

Chromosome painting probes (WCP)

Hybridize to whole chromosomes or regions

Characterize chromosomal structural changes in metaphase cells

Unique DNA sequence probes (LSI)

Hybridize to unique DNA sequences

Detect gene rearrangements, deletions, and amplifications

CENTROMERIC PROBES

(Ch 13 red, Ch18 pink, Ch 21 green, X yellow, Y white)

LOCUS-SPECIFIC PROBES

Ch 15 centromere (green)

Ch 15 PWS critical region (red)

CHROMOSOME PAINTING PROBES (CH 9 GREEN, DER CH 10)

CHROMOSOME PAINTING PROBES

MULTIPLEX LIGATION-DEPENDENT PROBE

AMPLIFICATION (MLPA)

• Gold standard for DNA copy number quantification

• Also be applied to investigate the methylation status of DNA sequences

• Popular applications include:

• Predisposition to Cancer

• Neuromuscular Disorders

• Intellectual Disability

• Solid Tumours

DETECTION OF X CHROMOSOME COPY

COMPARATIVE GENOMIC HYBRIDIZATION (ARRAY CGH)

CHROMOSOMAL ABNORMALITIES WITH ARRAY CGH

COMPARISON BETWEEN DIFFERENT METHODS FOR THE DETECTION OF GENE DELETIONS/DUPLICATIONS

POLYMERASE CHAIN REACTION (PCR)

USES FOR PCR

Research

Gene cloning

Real-time PCR

DNA sequencing

Clinical

DNA fingerprinting

Crime scene analysis

Paternity testing

Archeological finds

Genetically inherited diseases

RFLP (RESTRICTION FRAGMENT LENGTH POLYMORPHISMS)

• RFLP is an enzymatic procedure for separation and identification of desired fragments of DNA.

• Using restriction endonuclease enzymes fragments of DNA is obtained and the desired fragment is detected by using restriction probes.

• May be used to differentiated two organism by analysis of patterns derived from cleavage of their DNA.

• Variations commonly result in RFLPs:

• Single-base changes in the nucleotide sequences (SNP),

• Tandem repeats (VNTR),

• Polymorphisms,

• Mutations.

• SNP or VNTR, are simply markers, which, in most cases, have no known effect on the structure or rate of production of any particular protein.

APPLICATION OF RFLP TEST

Paternity test

Criminal investigation

To detect mutated gene

QUANTITATIVE REAL TIME PCR

QRT-PCR ANALYSIS

WESTERN BLOTTING

WB RESULTS

DNA SEQUENCING AND ITS TYPES

?

DNA extraction PCR

Gel electrophoresis

Insect identification

ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCA

DNA sequencing

Bioinformatics

DNA SEQUENCING

Probably the most important technique available to the molecular biologist is DNA Sequencing:

The precise order of nucleotides in a piece of DNA can be determined.

The DNA sequencing methods have been more than 40 years:

Since the mid-1970s rapid and efficient sequencing has been possible.

USE OF SEQUENCING

• The sequence of specific and single genes

• Larger genetic regions (i.e. clusters of genes or operons)

• Full chromosomes

• Entire genomes

Researchers in: molecular biology or genetics

Medical personnel: treatment, genetic counseling

DNA SEQUENCING

•Basic methods

Maxam-Gilbert sequencing (based on chemical modification of DNA and subsequent cleavage at specific bases)

Chain-termination methods (This method developed by Frederick Sanger and coworkers in 1977)

•Advanced methods

Shotgun sequencing (DNA sequences longer than 1000 base pairs,

DNA to be broken into random fragments. )

Bridge PCR (fragments are amplified upon primers attached to a solid surface)

High-throughput methods (NGS)

Chemical sequencing

Sanger sequencing

Developed by Allan Maxam and Walter Gilbert in 1976–1977.

This method uses double-stranded DNA samples

Performed by chain breakage at specific nucleotides.

Sequences DNA fragments containing upto ~500 nucleotides in length

A T

DMS

G

G G

G

FA

G A

G G

A G

A

H

C T

T C

T C

C

H+S

C C

C

C

MAXAM-GILBERT SEQUENCING

CHEMICAL MODIFICATION AND CLEAVAGE

Base Modification using Dimethyl sulphate

Purine

Adenine

Guanine

Only DMS------- G

DMS+ Formic acid-------G+A

Base modification using Hydrazine

Pyrimidine

Cytosine

Thymine

Hydrazine----- C+T

Hydrazine + NaCl--------C

Cleavage of Sugar Phosphate backbone using Piperidine

Sequencing gels are read from bottom to top (5′ to 3′).

G G+A T+C C

3′ A A G C A A C G T G C A G 5′

Longer fragments Shortest fragments

G

A

MAXAM-GILBERT SEQUENCING

Single stranded DNA template

A primer for DNA synthesis

DNA polymerase

Known as dideoxy sequencing method, use of analogue of normal

nucleotide 2’,3’-dideoxynucleoside triphosphates (ddNTPs).

The four different ddNTPs are labeled with different fluorescent

dyesfluorescent dyes

SANGER SEQUENCING

SANGER SEQUENCING: PROCESS

Get enough quantity of DNA (Run PCR)

Prepare PCR reaction mix as below:

Primer, Taq polymerase, template(ssDNA), dNTPS (All) and ddNTPs(ddATP , ddGTP,ddCTP & ddTTP respectively)

Run PCR

Perform Gel Electrophoresis

Interpret results

INTERPRETATION OF SEQUENCING CHROMATOGRAMS

SUBSTITUTION

INSERTION OR DELETION

PATHWAYS TOWARD HUMAN DISEASE GENE

IDENTIFICATION

LINKAGE ANALYSIS

NEXT-GENERATION SEQUENCING

Three general steps in NGS

1. Library preparation: libraries are created using random

fragmentation of DNA, followed by ligation with custom linkers

2. Amplification: the library is amplified using clonal amplification

methods and PCR

3. Sequencing: DNA is sequenced using one of several different

approaches

Gerald Goh and Murim Choi, 2012, Genomics & Informatics

Predicting the functional effect :

PlyPhen2 SIFT MutationTaster phyloP GERP++ dbNSFP …….

AVAILABLE NEXT-GENERATION SEQUENCING

PLATFORMS

Reversible terminator sequencing (Illumina)

Massively Parallel Signature Sequencing (MPSS)

Polony sequencing

454 pyrosequencing

Illumina (Solexa) sequencing

Sequencing by ligation (SOLiD)

Ion torrent semiconductor sequencing

DNA nanoball sequencing

Heliscope single molecule sequencing

Single molecule real time (SMRT) sequencing

.........

BRIDGE PCR

DNA fragments are flanked with adaptors.

A flat surface coated with two types of primers, corresponding to the

adaptors.

Amplification proceeds in cycles, with one end of each bridge tethered

to the surface.

Used by illumina/Solexa.

Fragments, with adaptors, are PCR amplified

within a water drop in oil.

One primer is attached to the surface of a bead.

Used by 454, Polonator and SOLiD.

EMULSION PCR

Rothberg and Leomon Nat Biotechnol. 2008

COMPARISON OF NEXT-GENERATION SEQUENCING

METHODS