DNA, DETECTION TYPES OF DNA

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

 Genomic DNA / Nuclear DNA:

 Comprises the 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

 ~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

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

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

A  Most are “silent” G  Intragenic  Promoters and other regulatory sequences  Introns  Exons  5’ and 3’ untranslated regions  Coding sequence (~1-2% of genome)

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 SILENT SEQUENCE CHANGE (SYNONYMOUS SNP)

mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln

Sequence mRNA variant A U G A A G U U U G G U G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln

Changes that do not alter the encoded amino acid Missense Mutation (Nonynonymous SNP)

mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln

mRNA Missense A U G A A G U U U A G C G C A U U G C A A Protein Met Lys Phe Ser Ala Leu Gln

Missense: changes to a codon for another amino acid (can be harmful mutation or neutral variant) Nonsense Mutation (Nonynonymous SNP)

mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln

mRNA A U G U A G U U U G G C G C A U U G C A A Nonsense Protein Met

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

Frameshift Mutations

mRNA Normal A U G A A G U U U G G C G C A U U G C A A Protein Met Lys Phe Gly Ala Leu Gln

mRNA Frameshift A U G A A G U U G G C G C A U U G C A A Protein Met Lys Leu Ala

Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein Splice-site Mutations

Exon 1 Intron Exon 2 Intron Exon 3

Exon 2

Exon 1 Exon 3 Altered mRNA

Splice-site mutation: a change that results in altered RNA sequence 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  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 FLUORESCENT IN SITU HYBRIDIZATION (FISH)

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

Interphase or metaphase cells on slide (in situ)

Probe

Microscopic signal (interphase) 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

• 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

 Clinical  Research  DNA fingerprinting  Gene cloning  Crime scene analysis

 Paternity testing  Real-time PCR  Archeological finds

 DNA sequencing  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 PCR Insect DNA extraction identification ?

Bioinformatics

DNA sequencing ACAGATGTCTTGTAATCCGGCCGTTG GTGGCATAGGGAAAGGACATTTAGT GAAAGAAATTGATGCGATGGGTGGA TCGATGGCTTATGCTATCGATCAATC AGGAATTCAATTTAGAGTACTTAAT AGTAGCAAAGGAGCTGCTGTTAGAG CAACACGTGCTCAGGCAGATAAAAT ATTATATCGTCAAGCAATACGTAGT ATTCTTGAATATCAAAAATTTTTGT TGGTTATTCA 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

 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) Chemical sequencing

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

MAXAM-GILBERT SEQUENCING

DMS FA H H+S

G G C C G A T C G G T G G C C C A T G C A C A T

 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 CHEMICAL MODIFICATION AND CLEAVAGE

 Base Modification using  Base modification using Dimethyl sulphate Hydrazine

 Purine  Pyrimidine

 Adenine  Cytosine

 Guanine  Thymine

 Only DMS------G  Hydrazine----- C+T

 DMS+ Formic acid------G+A  Hydrazine + NaCl------C

Cleavage of Sugar Phosphate backbone using Piperidine

MAXAM-GILBERT SEQUENCING

3′ A A G G+A T+C C G Longer fragments C A A A C G T Shortest fragments G C G A G 5′

Sequencing gels are read from bottom to top (5′ to 3′). SANGER 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: 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 Predicting the functional effect : PlyPhen2 SIFT MutationTaster phyloP GERP++ dbNSFP …….

Gerald Goh and Murim Choi, 2012, Genomics & Informatics 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. EMULSION PCR

Rothberg and Leomon Nat Biotechnol. 2008

 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. COMPARISON OF NEXT-GENERATION SEQUENCING METHODS