DNA SEQUENCING

GENOMIC ARRAY TECHNOLOGY

Simultaneous study of large numbers of targets (or samples)

GENOMIC ARRAY TECHNOLOGY

Arrays are applied to gene (DNA) amplification or deletion on comparative genome hybridization arrays and to gene-expression (RNA or protein) analysis on expression arrays

• Macroarrays

• Microarrays

• High-density oligonucleotide arrays

• Microelectronic arrays

approaches to array technology:

NEXT-GENERATION SEQUENCING

Also called as Massive Parallel Sequencing

NEXT-GENERATION SEQUENCING

Not done by students; capacity is very broad; used to know the whole sequence of the genome

NEXT-GENERATION SEQUENCING

Designed to sequence large numbers of templates carrying millions of bases simultaneously

• Pyrosequencing

• Reversible dye terminator sequencing

• Ion-conductance sequencing

• Single-molecule sequencing

• Sequencing by ligation

NGS technologies include the following:

Powerful computer data assembly systems

are required to organize the massive amounts of sequence information that are generated

NEXT-GENERATION SEQUENCING

requires strong computer support as well as terabytes of storage space to accommodate large raw data sets

DNA SEQUENCING

It is a molecular technique wherein we determine the exact sequences of the DNA molecule [exact bases or nucleotides present in the DNA molecule]

Direct Sequencing

able to know the exact bases of the DNA;

Direct Sequencing

Pyrosequencing

Bisulfate DNA Sequencing

RNA Sequencing

Next-Generation Sequencing

Types of sequencing

NEXT-GENERATION SEQUENCING

considered as the most specific and can sequence the whole genome; here, we are using super computers

DIRECT SEQUENCING

Most definitive molecular method to identify genetic lesions

DIRECT SEQUENCING

It is important to know the exact nucleotide or base in each DNA molecule to know if there’s gene mutation or etc.

MANUAL SEQUENCING AND AUTOMATED FLUORESCENT SEQUENCING

There are two approaches in direct sequencing, namely:

MANUAL SEQUENCING

Direct determination of the order, or sequence, of nucleotides in a DNA polymer

MANUAL SEQUENCING

Manual inspection of the result – we are using gel electrophoresis [manual inspection of the bands]

MANUAL SEQUENCING

Limited DNA strands to read; for short strands of DNA

Automated Fluorescent Sequencing

We make use of machines such as PCR or themocycler, capillary electrophoresis, etc. – much faster in sequencing and can sequence longer DNA strands

Automated Fluorescent Sequencing

Fluorescent dyes used for sequencing have distinct “colors,” or peak wavelengths of fluorescence emission, that can be distinguished by automated sequence

CHEMICAL (MAXAM-GILBERT) SEQUENCING

Developed by Allan M. Maxam and Walter Gilbert

- Earliest sequencing technique that was developed (early 1970s); during this time, they were able to sequence very short sequences.

CHEMICAL (MAXAM-GILBERT) SEQUENCING

Efficient way to determine short runs of sequence data - Cannot reach 1000+ bases since we are using gel electrophoresis

CHEMICAL (MAXAM-GILBERT) SEQUENCING

required a double- or single- stranded version of the DNA region to be sequenced, with one end radioactively labeled

methylates the guanine; all fragments with guanine will be left in this tube

tube 1 = dimethylsulfate

all fragments with purine will be left in this tube

tube 2 = formic acid

tube 3 = hydrazine:

all pyrimidine will be left in this tube

all cytosine will be left in this tube

tube 4 = hydrazine with salt:

MAXAM-GILBERT SEQUENCING

The sequence was inferred from the bands on the film

Guanine

Has band on the second and first lane

Adenine

has band on the second lane

Cytosine or thymine

Band on the third lane

Cytosine

Band on the fourth lane

MAXAM-GILBERT SEQUENCING

Disadvantage of this sequencing technique: exposed to hazardous chemical and only applicable for short sequences

Hydrazine and Piperidine

These are highly reactive chemicals used in maxam-gilbert sequencing

DIDEOXY CHAIN TERMINATION (SANGER) SEQUENCING

Modification of the DNA replication process

DIDEOXY CHAIN TERMINATION (SANGER) SEQUENCING

A short, synthetic, single-stranded DNA fragment just 5ʹ to the region of DNA to be sequenced

DIDEOXY CHAIN TERMINATION (SANGER) SEQUENCING

It is the same with how we use the PCR - It also uses template, primer, polymerase enzyme, etc

DIDEOXY CHAIN TERMINATION (SANGER) SEQUENCING

This is less hazardous since we are using modified dNTP = ddNTP

DIDEOXY CHAIN TERMINATION (SANGER) SEQUENCING

the 5’ end of the primer is labeled with fluorescent = 32P- or 35S-labeled deoxynucleotides [radioactive label or fluorescent dyes]

ddNTP [DIDEOXYNUCLEOTIDE]

Lacks the hydroxyl group found on the 3ʹ ribose carbon of the deoxynucleotides

dNTP

3’ carbon has hydroxyl group; only the 2’ carbon is deoxygenated

ddNTP

Since it is deoxygenated, there will be no formation of phosphodiester bond called chain termination

Chain Termination

DNA synthesis will stop upon incorporation of a ddNTP into the growing DNA chain

polymerization will terminate too frequently early along the template

If we Increase ddNTPs:

infrequent or no termination

if we decrease ddNTPs

POLYACRYLAMIDE GEL ELECTROPHORESIS

You can separate even with 1 base difference

POLYACRYLAMIDE GEL ELECTROPHORESIS

The products of each of the four sequencing reactions are loaded into adjacent lanes → Labeled A, C, G or T

POLYACRYLAMIDE GEL ELECTROPHORESIS

The fragment patterns are visualized by the signal on the 32Plabeled primer (or incorporated deoxynucleotide)

POLYACRYLAMIDE GEL ELECTROPHORESIS

All fragments from a given tube will end in the same ddNTP - Example: all the fragments synthesized in the ddCTP tube end in C

SEQUENCING LADDER

Easier to read if you have shorter DNA fragments

Cycle Sequencing and Automated Fluorescent Sequencing

Automated Methods of DNA Sequencing

CYCLE SEQUENCING

Modified Sanger Method

CYCLE SEQUENCING

The sequencing reaction took place in a thermal cycler

CYCLE SEQUENCING

Timed manual starting and stopping of the sequencing reactions were not necessary

CYCLE SEQUENCING

Using heat-stable enzymes

• With in vitro removal of the exonuclease activity

• Using double stranded template

AUTOMATED FLUORESCENT SEQUENCING

The cycle sequencing is the principle for this type of sequencing

AUTOMATED FLUORESCENT SEQUENCING

We are using fluorescent dyes to label [2 ang nil label: primer (5’ end) and ddNTP (3’ end)]

AUTOMATED FLUORESCENT SEQUENCING

Universal systems combined automation of DNA isolation of the template and setup of the sequencing reactions

Fluorescein

Rhodamine

Bodipy

Examples of Fluorescent dyes

Fluorescent dyes

used for sequencing have distinct “colors,” or peak wavelengths of fluorescence emission

GREEN

end color of adenine in Automated Fluorescent Sequencing

BLUE

end color of Cytosine in Automated Fluorescent Sequencing

YELLOW OR BLACK

end color of Guanine in Automated Fluorescent Sequencing

RED

end color of Thymine in Automated Fluorescent Sequencing

Capillary Electrophoresis

the automated method of electrophoresis

DYE PRIMER SEQUENCING

The label is on the 5’ end [the primer itself]

DYE PRIMER SEQUENCING

The dye molecules are attached covalently to the 5ʹ end of the primer

DYE TERMINATOR SEQUENCING

DYE TERMINATOR SEQUENCING

DYE PRIMER SEQUENCING

Primer id unlabeled • One of the four fluorescent dyes covalently attached to each of the ddNTPs

DYE TERMINATOR SEQUENCING

The color of the dye corresponds to the ddNTP that terminated the strand

DYE TERMINATOR SEQUENCING

The primers here are not labelled; the one that has a label are the ddNTPs itself.

• Subject to electrophoresis

COLUMN OR BEADS AND ETHANOL PRECIPITATION

Excess dye terminators are removed with:

SEQUENCING LADDER

The fragments formed are single-stranded; keep it as single stranded as possible -

• Add denaturing agents such as formamide or urea to prevent rehybridization

SEQUENCING LADDER

The ladders are heated to 95°C to 98°C for 2 to 5 minutes and placed on ice just before loading

ELECTROPHORESIS

The four sets of sequencing products in each reaction are loaded onto a single gel lane or capillary

ELECTROPHORESIS

The migrating fragments pass a laser beam and a detector in the automated sequencer

ELECTROPHEROGRAM

The one being read in the computer

ELECTROPHEROGRAM

For each nucleotide = 1 peak should be observed

ELECTROPHEROGRAM

The sequencing software reads, or “calls,” the bases from the smallest (fastest-migrating) fragments that first pass the detector to the largest based on the dye emission wavelength

ELECTROPHEROGRAM

is a series of peaks of the four fluorescent dyes as the bands of the sequencing ladder migrate by the detector

BASE CALLING

The computer itself will read/call the sequence or will identify the nucleotide base by checking the fluorescence color

BASE CALLING

is the process of identification of bases in a sequence by sequencing software

SEQUENCE INTERPRETATION

Interpretation of sequencing data from a dye terminator reaction depends on the quality of the electropherogram

Dye Blobs

Failure to clean the sequencing ladder properly can cause

Dye Blobs

bright flashes of fluorescence