molecular techniques-1(introduction).ppt file/molecular techniques-1... · 2014. 3. 10. · title:...

Molecular / Nucleic acid Molecular / Nucleic acid

techniques in diagnosistechniques in diagnosis

Professor Md. Akram HossainProfessor Md. Akram Hossain

Department of Microbiology, MMCDepartment of Microbiology, MMC

What is molecular techniques?What is molecular techniques?

��Handling of nucleic acids for diagnosis and Handling of nucleic acids for diagnosis and

other purposes are known as molecular other purposes are known as molecular

techniques (Nucleic acid based techniques).techniques (Nucleic acid based techniques).

Genetic Engineering -

2

�Genetic Engineering - the process of

isolating a gene from the DNA of one organism

and transferring it to the DNA of another

organism

Prof.Muhammad Akram Hossain, Introduction to Molecular techniques

Why Nucleic acid based techniques?Why Nucleic acid based techniques?

�� Diagnosis possible at most fundamental levels Diagnosis possible at most fundamental levels by information in DNAby information in DNA

�� Advantages of DNA as substrate for analysisAdvantages of DNA as substrate for analysis

3

�� Sturdy bioSturdy bio--molecule easy to handlemolecule easy to handle

�� Obtained from fresh or fixed tissue or blood Obtained from fresh or fixed tissue or blood specimenspecimen

�� Better than protein for handlingBetter than protein for handling


What are Nucleic Acids? Why so What are Nucleic Acids? Why so called?called?

� Acids originally derived from nucleus

– DNA – Deoxy ribonucleic acid

– RNA – Ribonucleic acid

©2001 Timothy G. Standish

– RNA – Ribonucleic acid

� The double stranded DNA serves as the genetic

material for all living organisms

� RNA also serves as genetic materials in viruses

Why NA serves as genetic materialsWhy NA serves as genetic materials

� The structure of nucleic acids reveals both why they are excellent moleculesfor information storage and transmission

� The monomers joined together to make nucleic acids are like letters in the English language, nothing prevents the letters from


letters in the English language, nothing prevents the letters from being arranged in any sequence to create an almost infinite set of words, sentences, paragraphs etc.

� Because nucleic acids are made up of subunits whose sequence is uninfluencedby chemical interactions between the subunits they excel as information storage molecules

Structure and Function of Structure and Function of Genetic MaterialGenetic Material

�DNA & RNA

�DNA=deoxyribonucleic acid

�RNA=ribonucleic acid


�Basic building blocks:

�Nucleotides

�Phosphate group

�Pentose sugar –Ribose or Deoxyribose

�Nitrogenous base – Purine /pyrimidine

H

A NucleotideA NucleotideAdenosine Mono Phosphate (AMP)Adenosine Mono Phosphate (AMP)

NH2

N N

BaseP

O

OH

HO OPhosphate

H+

-


OH

OCH2

Sugar

H

HH

OH

N

N

N

O

2’3’

4’

5’

1’Nucleotide

Nucleoside

H

Pyrimidines

N

N

Adenine

N

N

NH2

Purines

Uracil(RNA)CH3

N ON

O

NH

N ON

O

NH

Thymine(DNA)

NH2

O

N

N NH

N

Guanine

N N

N O

NH2

N O

NH2

NCytosine

N ON N ON

+-

Base PairingBase PairingGuanine And CytosineGuanine And Cytosine

-+

+ -

- Thymine+

Adenine

Base PairingBase PairingAdenine And ThymineAdenine And Thymine

+-

Base PairingBase PairingAdenine And CytosineAdenine And Cytosine

+

-

-

Base PairingBase PairingGuanine And ThymineGuanine And Thymine

-

+

+

Structure of DNAStructure of DNA

� Each molecule composed two strands, coiled into double helix

� Two strands held together


� Two strands held together by hydrogen bonds between bases (A- T, G –C)

� Strands are complimentary due to appropriate pairing between bases

Special features of DNA StructureSpecial features of DNA Structure

1. Strong covalent bond binds them within the strand,

2. Weak bonds between strands, can be separated easily by heat or alkali –


separated easily by heat or alkali –denaturation.

3. Spontaneous repairing occurs at physiological condition (Temp, PH)

4. This is the basis for many of the molecular techniques

P

O

HO O

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NHN

NH

N

N

DDNNAA

H

O

OH

H OH

P

O

HO

O

O

CH2

H2O

5’Phosphate group3’Hydroxyl group


H

OCH2

HOH

P

O

O

HO

O

O

CH2

NH2NN

N O

NH2

N

AAOH

P

O

HO

O

O

CH2

HO

H

P HO

O

O

CH2

O

H2O

3’Hydroxyl group

5’Phosphategroup

Because of specific base paring, any single stranded sequence of DNA or RNA can be used as a template for production of the complimentary strand

The Watson The Watson -- Crick Crick Model Of DNAModel Of DNA

3.4 nm1 nmMinor

groove

T AG C

C G

G CT A

A TC G -

--

-

--

-

-

--

-

-

---

-


0.34 nm

Majorgroove

A TC G

C GG C

T A

A T

--

--

--

--

--

--

--

--

Forms of the Double HelixForms of the Double Helix

2.8 nmMinorgroove

1.2 nm

A DNA

1 nmMinorgroove

T AG C

C G

G CT A

A TC G

3.9 nm

B DNA

6.8 nm

0.9 nm

Z DNA


0.26 nm

Majorgroove

Majorgroove

A TC G

C G

G CT A

A T

0.34 nm

+32.7o Rotation/Bp11 Bp/turn

-30.0o Rotation/Bp12 Bp/turn

+34.6o Rotation/Bp10.4 Bp/turn

0.57 nm

� C-DNA:– Exists only under high dehydration conditions– 9.3 bp/turn, 0.19 nm diameter and tilted bases

� D-DNA:– Occurs in helices lacking guanine– 8 bp/turn

E-DNA:

Even More Forms Of DNAEven More Forms Of DNA

BB--DNA appears to be the DNA appears to be the most common form most common form in vivoin vivo. . However, under some However, under some circumstances, alternative circumstances, alternative


� E-DNA:– Like D-DNA lack guanine– 7.5 bp/turn

� P-DNA:– Artificially stretched DNA with phosphate groups found inside the long

thin molecule and bases closer to the outside surface of the helix– 2.62 bp/turn

circumstances, alternative circumstances, alternative forms of DNA may play a forms of DNA may play a biologically significant biologically significant role.role.

SupercoilingSupercoiling

Opened negatively supercoiled

DNA

Open circle DNA with no supercoiling


Negatively (twisting to the

left) supercoiled

DNA

Opening negatively supercoiled DNA may contribute to strand separation

P

O

-O O

H

P

O

O-

-O

O

O

CH2

NH2

N

N

N

N

O

O

NHN

H O

H OH

P

O

O-

O

O

CH2

Distribution Of Negative Charge Prevents Distribution Of Negative Charge Prevents DNA AnnealingDNA Annealing

H

OCH2

HOH

P

O

O

-O

O

O

CH2

O

NH2N

NH

N

N O

NH2

N

OH

P

O

O

O

CH2

P O-

O

O

CH2

O

O-

O-

NaCl

P

O

-O O

H

P

O

O-

-O

O

O

CH2

NH2

N

N

N

N

O

O

NHN

H O

H OH

P

O

O-

O

O

CH2NaCl

Cl-

Na+

Salts Allow DNA AnnealingSalts Allow DNA Annealing

H

OCH2

HOH

P

O

O

-O

O

O

CH2

O

NH2N

NH

N

N O

NH2

N

OH

P

O

O

O

CH2

P O-

O

O

CH2

O

O-

O-

Na+

Cat ions can cancel out the negative charge carried on the sugar phosphate backbone.

Na+

Na+

Na+

Na+P

O

-O O

H

P

O

O-

-O

O

O

CH2

NH2

N

N

N

N

O

O

NHN

H O

H OH

P

O

O-

O

O

CH2


Na+

Na+

Na+

Na+H

OCH2

HOH

P

O

O

-O

O

O

CH2

O

NH2N

NH

N

N O

NH2

N

OH

P

O

O

O

CH2

P O-

O

O

CH2

O

O-

O-

Na+

Na+

Na+

H O

H OH

P

O

O-

O

O

CH2


P

O

-O O

H

P

O

O-

-O

O

O

CH2

NH2

N

N

N

N

O

O

NHN

Na+

Na+

Na+

Na+

Na+

Na+

OH

P

O

O

O

CH2

P O-

O

O

CH2

O

O-

O-

H

OCH2

HOH

P

O

O

-O

O

O

CH2

O

NH2N

NH

N

N O

NH2

N

Na+

DNATranscription

The Central Dogma of Molecular BiologyThe Central Dogma of Molecular Biology

Cell


mRNA

Polypeptide(protein)

TranslationRibosome


transcription translationDNA PROTEINRNA

Transcription initiation and elongationTranscription initiation and elongation

1. Genes need to be expressed to be “genes”

2. Transcription is directed to specific


directed to specific locations (promoters)

3. RNA is elongated in the 5’-to-3’ direction

Key points to remember about translationKey points to remember about translation

2. Protein is produced from the N-terminus to the C-terminus; the mRNA 5’ end

1. Translation begins at AUG, ends at stop codon (UAG, UAA, UGA)


terminus; the mRNA 5’ end encodes the N-terminus of the protein

3. Prokaryotes : initiation near Shine-Dalgarno sequence

Eukaryotes : ribosome scanning to first AUG

DNA RNA

deoxyribose

ll-stranded

A, G, C, T

A-T base pairing

ribose

l-stranded

A, G, C, U

A-U base pairing


A-T base pairing A-U base pairing

DNA

RNA

TRANSCRIPTION


Transcription


Click to view animation.

Translation: Protein SynthesisTranslation: Protein Synthesis


Applications of molecular biologyApplications of molecular biology

�� DiagnosisDiagnosis

�� Microbiological diseasesMicrobiological diseases

�� Detection of nucleic acid sequences of pathogens in samplesDetection of nucleic acid sequences of pathogens in samples

�� Diagnosis of genetic disordersDiagnosis of genetic disorders

�� Diagnosis of neoplasmsDiagnosis of neoplasms

32

�� Diagnosis of neoplasmsDiagnosis of neoplasms

�� Forensic medicineForensic medicine

�� Genomic identification of forensic samples (DNA Lab at DMC)Genomic identification of forensic samples (DNA Lab at DMC)

�� Genetic engineeringGenetic engineering

�� Vaccine productionVaccine production

�� Insulin & other hormonesInsulin & other hormones

�� InterferonInterferon

�� ResearchResearchProf.Muhammad Akram Hossain,

Introduction to Molecular techniques

Outline of the NA technologyOutline of the NA technology

�� Sample Sample ––

�� Blood, tissue, secretions, body fluids etcBlood, tissue, secretions, body fluids etc

�� Target Nucleic acidTarget Nucleic acid

�� ProceduresProcedures

33

�� ProceduresProcedures

�� HybridizationHybridization

�� Target amplificationTarget amplification

�� Detection of amplified productDetection of amplified product


Nucleic acid probes Nucleic acid probes

�� Are strands with labeled known sequence of NA to detect strands Are strands with labeled known sequence of NA to detect strands

with complementary base sequencewith complementary base sequence

�� DNA probesDNA probes

�� Single DNA strands upto 100 bases prepared by atutomated Single DNA strands upto 100 bases prepared by atutomated

34

�� Single DNA strands upto 100 bases prepared by atutomated Single DNA strands upto 100 bases prepared by atutomated

sinthesizerssinthesizers

�� Large DNA sequences by cloning in bacteriaLarge DNA sequences by cloning in bacteria

�� RNA probesRNA probes

�� Prepared by cloning corresponding DNA sequences using as Prepared by cloning corresponding DNA sequences using as

template for intemplate for in--vitro transcription i.e producing a vitro transcription i.e producing a

complimentary RNA strand from template DNAcomplimentary RNA strand from template DNAProf.Muhammad Akram Hossain, Introduction to Molecular techniques

DNA &RNA probesDNA &RNA probes

�� The probes can easily be tagged with radioisotopes, The probes can easily be tagged with radioisotopes, flurochromes or enzymatic markers & acts as a flurochromes or enzymatic markers & acts as a molecular stainmolecular stain

�� Probes possess an extraordinary degree of specificityProbes possess an extraordinary degree of specificity

35

�� Probes possess an extraordinary degree of specificityProbes possess an extraordinary degree of specificity

�� A typical probe is capable of recognizing and A typical probe is capable of recognizing and binding selectively to single copy of its binding selectively to single copy of its complimentary sequence among 3 billion base pairs complimentary sequence among 3 billion base pairs in human genome.in human genome.


PrimersPrimers

�� PrimersPrimers

�� Short 20Short 20--30 nucleotides with sequences 30 nucleotides with sequences

complimentary to the amplicon ?complimentary to the amplicon ?

�� Used in pairs, they define the endpoints of the Used in pairs, they define the endpoints of the

36

�� Used in pairs, they define the endpoints of the Used in pairs, they define the endpoints of the

amplified regionamplified region

�� Usually designed such thatUsually designed such that

Tm between 55Tm between 55°° and 72and 72°°CC

3’ ideally with a high GC content3’ ideally with a high GC content


molecular techniques-1(introduction).ppt file/molecular techniques-1... · 2014. 3. 10. · title:...

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