molecular techniques department of stem cells & regenerative medicine acecr

Molecular Techniques

Department of Stem Cells & Regenerative Medicine

ACECR

DNA & RNA

•DNA is a nucleic acid, made of long chains of nucleotides

Nucleotide

Phosphate group

Nitrogenous base

Sugar

Polynucleotide Sugar-phosphate backbone

DNA nucleotide

Phosphategroup

Nitrogenous base(A, G, C, or T)

Thymine (T)

Sugar(deoxyribose)

DNA and RNA are polymers of nucleotides

Bases

• Adenine double ringed =

• Guanine

• Thymine single ringed =

• Cytosine

purines

pyrimidines

Hydrogen bonds between bases hold the strands together: A and T, C and G

Untwisting and replication of DNA

• each strand is a template for a new strand

helicase

5 end

Parental DNA

DNA polymerasemolecule

53

35

35

Daughter strandsynthesizedcontinuously

Daughter strandsynthesizedin pieces

RNA is also a nucleic acid

– different sugar

– U instead of T

– Single strand, usuallyNitrogenous base

(A, G, C, or U)

Uracil (U)

Sugar(ribose)

DNA molecule

Gene 1

Gene 2

Gene 3

DNA strand

TRANSCRIPTION

RNA

Polypeptide

TRANSLATIONCodon

Amino acid

DNA of gene

PromoterDNA

RNA polymerase

Elongation

Termination

TerminatorDNACompleted RNA

RNApolymerase

GrowingRNA

U C A G

U

C

A

G

GACU

GACU

GACU

GACU

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

phe

leu

leu

ile

met (start)

val

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

ser

pro

thr

ala

UAUUACUAAUAG

CAUCACCAACAG

AAUAAC

AAGAAA

GAUGACGAAGAG

tyr

stopstop

his

gln

asn

lys

asp

glu

UGUUGCUGAUGG

CGUCGCCGACGG

AGUAGCAGAAGG

GGUGGCGGAGGG

cys

stoptrp

arg

ser

arg

gly

Firs

t B

ase

Third

Base

Second Base

Virtually all organisms share the same genetic code “unity of life”

Techniques molecular biology Polymerase chain reaction (PCR)

Gel electrophoresis Macromolecule blotting and probing

Southern blotting Northern blotting Western blotting

Polymerase Chain Reaction

Polymerase Chain Reaction

Invented by Kary Mullis and his colleagues in the 1983.

Nobel prize 1993.

PCR Reaction Components

Template: previously isolated and purified.

Two primers: to flank the target sequence)18-28 nucleotides).

Four deoxynucleosides triphosphate (dNTPs) : to provide energy and nucleosides for the synthesis of DNA.

Buffer system containing magnesium.

DNA polymerase.

Thermal Cycler

PCR Cycles

Denaturation

Annealing

Elongation

As amplification As amplification proceeds , the DNA proceeds , the DNA

sequence between primers sequence between primers doubles after each cycles doubles after each cycles (The amplification of the (The amplification of the

target sequence target sequence proceeding in an proceeding in an

exponential fashionexponential fashion ( 1 ( 1 2 4 8 2 4 8

16…………….)- up to 16…………….)- up to million of times the million of times the

starting amount until starting amount until enough is present to be enough is present to be

seen by gel seen by gel electrophoresis.electrophoresis.

Agarose Gel Electrophoresis

Since PCR amplifications can generate microgram

quantities of product, amplified fragments can

be visualized easily following staining with a

chemical stain such as ethidium bromide.

Variants of PCR

Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR. Quantitative PCR. ….

Polymerase Chain Reaction: Uses

The polymerase chain reaction (PCR) is a technique widely used in:

Molecular biology, Microbiology,

Genetics, Diagnostics clinical laboratories,

Forensic science, Environmental science,

Hereditary studies, Paternity testing, and

Many other applications…

Polymerase Chain Reaction clearly has the potential to become the routine laboratory method for diagnosis of a variety of human disorders :

Infectious Diseases: One area where the PCR technique will undoubtedly become a routine

method, is the detection of infectious agents, such as pathogenic bacteria, viruses or protozoa.

Cancer: Detection of malignant diseases by PCR. Recurrence of hematological cancers has also been evaluated. Detection of micro-metastasis in blood, lymph nodes and bone

marrow.

Inherited disorders diagnosed using PCR protocols:

Alpha1 antitrypsin B thalassaemia Cystic fibrosis Duchenne muscular dystrophy Myotonic dystrophy Haemophilia A and B Huntington`s chorea Phenylketonuria Sickle cell anaemia Familial adenomatous polyposis

Comparison of Southern, Northern, and Western analyses

‘Southern’ hybridization named after Sir Edwin Southern

Developed in 1975

Southern Blotting

Example – Looking for Gene X

Cell line 12 copies of Gene X

extract

DNA

Example – Looking for Gene X

Cell line 2

extract

DNA

Step 1. Restriction Enzyme Digestion

EcoR I EcoR I EcoR I EcoR I

Step 1. Restriction Enzyme Digestion

Step 2. Gel Electrophoresis

_ +

Step 2. Gel Electrophoresis

_ +

T G A A TC

A C AT T G

Step 3. DNA Denaturation

Eliminate hydrogen bonds with sodium hydroxide (NaOH)

Step 4. Transfer DNA to Membrane

Step 5. Making a Probe

A probe is a small (25-2000 bp) length of DNA or RNAComplementary to the sequence (gene) of interestLabeled for subsequent detection procedures

Step 6. Pre-hybridization

Prehybridization bufferscontain ‘blocking reagents’that occupy available binding sites on the membrane

Step 7. Hybridization

Step 7. Hybridization

Step 8. Washes

Step 9. Anti-DIG

Step 9. Anti-DIG

Step 10. Washes

Step 11. CSPD

Step 12. Detection

• DIG-labeled probes emitting minute amounts of light (chemiluminescence)

• 32P-labeled probes emitting ß-particles

Step 12. Detection

• DIG-labeled probes emitting minute amounts of light (chemiluminescence)

• 32P-labeled probes emitting ß-particles

• Autoradiography film can detect this radiation

Example – Looking for Gene X

Cell line 2

extract

DNA

3 Copies

Detection of the sickle-cell globin gene by Southern blotting

The flow chart of Northern hybridization

Prepare RNA samples and run RNA gel

Northern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

Any Question ?