dr. nasser a. elhawary prof. of medical genetics

Dr. Nasser A. ElhawaryProf. of Medical Genetics

DNATechnology

DNACLONING

Methods of DNA Analysis

DNA CLONING

Cloning of DNA segments:

results in the production of a

large no. of identical DNA

molecules from a common

ancestor.In vivo cell-based DNA Cloning.

In vitro PCR.

Dolly was cloned by Ian Wilmut & Keith Campbell in 1996

Dolly (5 July 1996 – 14 February 2003) was a female sheep, and the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer

EcoRI enzyme cuts the dsDNA segment into 2 sticky fragments)

Background >>> Restriction Enzymes…

Restriction endonucleases (RE): Enzymes that recognize a specific base sequence in a DNA segment and cleave or nick the DNA at this site

- - C C C G G G - -

- - G G G C C C - -

SmaI

Blunt ends

Enzyme Organism Cleavage site5` 3`

BamHI Bacillus amyloliquefaciens H G • G A T C C

EcoRI Escherichia coli RY 13 G • A A T T C

HaeIII Haemophillus aegyptius G G • C C

HindIII Haemophillus influenzae Rd A • A G C T T

HpaI Haemophillus parainfluenzae G T T • A A C

SmaI Serratia marcescens C C C • G G G

SalI Streptomyces albus G G • T C G A C

Examples of Restriction endonucleases with their recognition sequence and cleavage sites

Background >>> Vectors…

There are 5 main types of vectors

commonly used which include

plasmids, bacterio-phages,

cosmids, bacterial (BACs) and

yeast artificial chromosomes

(YACs)

1- In vivo cell based DNA cloning…

Basic steps of in vivo DNA cloning:

1. Generation of DNA fragments (using RE):

2. Recombination of DNA fragments (DNA

ligase).

3. Vector: (A self-replicating DNA that is used to transfer foreign DNA segments between host cells).

The incorporation of the target DNA into a vector allows the production of large amounts of that DNA.

4. Transformation of the host organism:

- The recombinant vector is introduced into specially modified bacterial host cells (Transformation process) (using potassium chloride).

- If transformed cells multiply → large quantities of identical copies of the original single target DNA or clones.

- The choice of vectors depends on a no. of factors, e.g. particular RE, the size of target DNA inserted.

1- In vivo cell based DNA cloning…

5. Screening for recombinant vectors.6. Selection of specific clones.

What is DNA Library? It is the collection

of recombin-ant of DNA molecules

generated from a specific source, e.g.

genomic or cDNA library.GeneBank: It is collection of clones that contains all the genetic information in an individual.

1- In vivo cell based DNA cloning…

DNA Cloning…Summary

DNA Cloning… Summary

Kary Mullis, 1980; Nobel Prize 1993

PCR is a cell-free DNA cloning. In 1985, PCR is a technique for

amplifying a short segment of DNA (of order >106) in an in vitro reaction.

It includes a three-step cycles. For 30 cycles, the dsDNA will give a PCR product of 230 (>109 copies).

PCR-steps:1- Denaturing 2- Annealing 3-

Extension

2- In vitro Polymerase Chain Reaction

2- In vitro PCR…

2- In vitro PCR…

RT-PCR (Reverse Transcription PCR): is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA.

The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.

RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript.

Thermal Cycler Systems

Gel Electrophoresis

Separate DNA fragments by their sizes (MW).

Apply an electric field.

DNA is -vely charged.

Separate through gel matrix.

Visualise DNA fragments under UV light.

Gel Electrophoresis of PCR products…

Vertical Gel Unit

To separate very close DNA fragments (1 bp) on Polyacrylamide gel (PAGE)

Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers

Transfer of

PCR to

ELISA

plate

NANODROP TECHNOLOGY

Quantitative DNA

Concentration

PCR Applications

• DNA Amplifications

• Detection of disease mutations (using RE,

ASO, SSCP, Denaturing HPLC, etc.)

• DNA sequencing (detect base-sequences)

• Gene expression studies

• Forensic medicine

RFLP: is Variations in the length of DNA frag-ments generated by a Restriction Endonuclease (RE).

Genetic variations almost never represent detectable differences that are functionally significant.

Polymorphism is a form of a genetic variation due to the presence or absence of a cutting site.

RFLP in 1970

Example of Genotyping (RFLPs)

IVS10 mutation in PAH gene

Jefffreys (1980) used a collection of probes that

simultaneously detect variability in short, repeated

DNA sequences at 8 to 15 loci scattered on different

chromosomes.

The resulting pattern of 25 to 60 bands is unique to

each individual (except for identical twins).

DNA Fingerprints describe this RFLP pattern of DNA

fragments (forensics).

Samples from blood, semen, hair, skin and tissues.

DNA Fingerprinting…

DNA Fingerprinting

DNA Restriction enzymedigest

+

-direction

ofmigration

Single Strand Conformational Polymorphism (SSCP)

Examples of DNA Hybridization & Probes

Allele-Specific Oligonucleotide (ASO):

Distinguishing between two DNA molecules differing by one base (N, M) by hybridization

1. Hybridization will allow the ‘perfect match’ ASO to hybridize to its target.

2. ASO with a single nucleotide with ‘mismatch’ will not hybridize.

Hybridization & Probes…

ASO-Dot Blot…

It indicates that labeled DNA can be used to monitor the expression of DNA molecules attached to a solid support.

It is defined as a high-throughput and versatile technology used for parallel gene expression analysis for thousands of genes of known and unknown function, to detect polymorphisms & mutations in gDNA.

Microarray Technology…

Microarray Chip…

Microarray Technology…

Patrick Brown at Stanford University 1990

Microarray Chip…

Glass cDNA microarray produced by using high-speed precision robot. This type of DNA microarray can bear between 10,000-20,000 spots (genes) on an area of 3.6 cm2.

Advanced DNA Technology

Real-Time PCR: TaqMan™ and Light cycler™ use fluorescent tech to detect mutations by allelic PCR products. Ingradients: DNA, primers, probes, dNTPs, Buffers, Taq enzymes.

Denaturing HPLC (DHPLC). DNA Sequencing. Next Generation Sequencing

(NGS).

DHPLC technology…

DHPLC-WAVE system. The columnSep separate the Heteroduplexes generating from a PCR product (a patient and wild type) at a critical temperature.

It uses a polymer separating column, UV to detect PCR products.

Determination of nucleotide sequence.

Technique invented in 1970’s.

F. Sanger - Termination method.

Dideoxynucleotides.

DNA Sequencing

Dideoxynucleotide

No OH-group at 3’-endprevents strand extension

CH2O

OPPP5’

3’

BASE

DNA Sequencing…

• It is the ‘Gold standard’ to screen mutation using the dideoxy chain termination method.

• Denatured PCR (ssDNA) forms a cDNA using primers in a PCReaction.

• ddNTP’s labeled with fluorescent dyes (4 colors) are used to terminate the PCR reaction.

• The reaction products are separated on PAGE or capillary electrophoresis.

• Identify from 400-1000 basepairs.

In 1970

Sanger sequencing reaction

• Single stranded DNA is amplified

in the presence of fluorescently

labeled ddNTPs that serve to

terminate the reaction and label

all the fragments of DNA

produced.

• The fragments of DNA are then

separated via polyacrylamide gel

electrophoresis and the sequence

read using a laser beam.

DNA Sequencing…

Shotgun Sequencing

• DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads.

• Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing.

• Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.

• Shotgun sequencing was one of the precursor technologies that was responsible for enabling full genome sequencing.

It is a top from 1995-2005

Next-Generation Sequencing (NGS)

• Although ‘shotgun sequencing’ was the most advanced technique for sequencing genomes from about 1995–2005, NGS have surfaced.

• NGS produces shorter reads (anywhere from 25–500bp) but many hundreds of thousands or millions of reads in a relatively short time (on the order of a day).

• This results in high coverage, but the assembly process is much more computationally expensive.

• NGS technology are vastly superior to “shotgun sequencing” due to the high volume of data and the relatively short time it takes to sequence a whole genome.

• The major disadvantage of ‘NGS’ is that the accuracies are usually lower (although this is compensated for by the high coverage).