NUCLEIC ACIDSNUCLEIC ACIDSSem I, 2011/2012Sem I, 2011/2012

Khadijah Hanim bt Abdul RahmanKhadijah Hanim bt Abdul Rahman

School of Bioprocess Eng, UniMAPSchool of Bioprocess Eng, UniMAP

Week 12: 30/11 & 1/12/2011Week 12: 30/11 & 1/12/2011

khadijahhanim@unimap.edu.mykhadijahhanim@unimap.edu.my

Learning OutcomesLearning Outcomes

DISCUSS basic structures, properties and DISCUSS basic structures, properties and functions of nucleic acids. functions of nucleic acids.

DISCUSS DNA isolation methodsDISCUSS DNA isolation methods

DefinitionsDefinitions

DNA stands for deoxyribonucleic acid. It is DNA stands for deoxyribonucleic acid. It is the genetic code molecule for most the genetic code molecule for most organisms.organisms.

RNA stands for ribonucleic acid. RNA RNA stands for ribonucleic acid. RNA molecules are involved in converting the molecules are involved in converting the genetic information in DNA into proteins. genetic information in DNA into proteins. In retroviruses, RNA is the genetic In retroviruses, RNA is the genetic material.material.

DNA structure : Watson and CrickDNA structure : Watson and Crick Watson and Crick 1953 1Watson and Crick 1953 1stst

proposed the proposed the double helixdouble helix as 3-D as 3-D structure of DNAstructure of DNA

Two polynucleotide chains wind Two polynucleotide chains wind around a common axis to form a around a common axis to form a double helixdouble helix. .

The two strands of DNA are The two strands of DNA are antiparallelantiparallel, but each forms a , but each forms a right-handed helix. right-handed helix.

The bases occupy the core of the The bases occupy the core of the helix and sugar-phosphate chains helix and sugar-phosphate chains run along the periphery, thereby run along the periphery, thereby minimizing the repulsions between minimizing the repulsions between charged phosphate groups.charged phosphate groups.

DNADNA

Consists of 2 polynucleotide strands wound around each Consists of 2 polynucleotide strands wound around each other to form a right-handed double helixother to form a right-handed double helix

Each nucleotide monomer in DNA is composed of:Each nucleotide monomer in DNA is composed of:- Nitrogenous base (purine @ pyrimidine)Nitrogenous base (purine @ pyrimidine)- Deoxyribose sugar (pentose, 5C)Deoxyribose sugar (pentose, 5C)- PhosphatePhosphate Mononucleotides are linked to each other by Mononucleotides are linked to each other by 3’,5’- 3’,5’-

phosphodiester bondsphosphodiester bonds These bonds join the These bonds join the 5’-hydroxyl group of the 5’-hydroxyl group of the

deoxyribose of 1 nucleotide to the 3’-OH group of the deoxyribose of 1 nucleotide to the 3’-OH group of the sugar unit of another nucleotidesugar unit of another nucleotide thru a thru a phosphatephosphate group. group.

PENTOSE SUGARPENTOSE SUGAR

In ribonucleotides, the In ribonucleotides, the pentose is ribosepentose is ribose

In deoxyribonucleotide (or In deoxyribonucleotide (or deoxynucleotides) the deoxynucleotides) the sugar is 2’-deoxyribose – sugar is 2’-deoxyribose – the carbon at position 2’ the carbon at position 2’ lacks a hydroxyl grouplacks a hydroxyl group

Nucleic acid structureNucleic acid structure

The antiparallel The antiparallel orientation of the 2 orientation of the 2 polynucleotide strands polynucleotide strands allows allows H bond to form H bond to form between nitrogenous between nitrogenous bases that are bases that are oriented toward the oriented toward the helix interiorhelix interior..

There are 2 types of base pairs (bp) in DNA:There are 2 types of base pairs (bp) in DNA:- AdenineAdenine (A- purine) pairs with (A- purine) pairs with thyminethymine (T- (T-

pyrimidine)- 2 hydrogen bondspyrimidine)- 2 hydrogen bonds- GuanineGuanine (G- purine) pairs with (G- purine) pairs with cytosinecytosine (C- (C-

cytosine)- 3 hydrogen bondscytosine)- 3 hydrogen bonds

If 1 strand has the base sequence If 1 strand has the base sequence AGGTCCG, so the other strand must have AGGTCCG, so the other strand must have sequence TCCAGGCsequence TCCAGGC

These hydrogen bonding interactions, a These hydrogen bonding interactions, a phenomenon known as phenomenon known as complementary complementary base pairingbase pairing, , result in the specific result in the specific association of the two chains of the double association of the two chains of the double helix.helix.

The overall structure of DNA The overall structure of DNA resembles a twisted staircase.resembles a twisted staircase.

The Dimension of crystalline DNA The Dimension of crystalline DNA have been precisely measured :have been precisely measured :

1) one turn of double helix span 1) one turn of double helix span 3.4nm and consist 10.4 base 3.4nm and consist 10.4 base pairs.pairs.

2) diameter of double helix is 2) diameter of double helix is 2.4nm- interior space of double 2.4nm- interior space of double helix- suitable for base-pairing helix- suitable for base-pairing purine-pyrimidine. purine-pyrimidine.

3) distance between adjacent 3) distance between adjacent base pairs is 0.34nm. base pairs is 0.34nm.

Noncovalent bonding Noncovalent bonding that contribute to the stability that contribute to the stability of DNA helicalof DNA helical structure :structure :

1) Hydrophobic interactions1) Hydrophobic interactions. The base ring . The base ring ππ cloud cloud of electrons between stacked purine & pyrimidine of electrons between stacked purine & pyrimidine bases is nonpolar. The clustering of bases bases is nonpolar. The clustering of bases component of nucleotide within double helix component of nucleotide within double helix stabilize structure, because it minimize their stabilize structure, because it minimize their interaction with water.interaction with water.

2) Hydrogen bond2) Hydrogen bond-between nucleotides.Base pairs, -between nucleotides.Base pairs, on close approach form hydrogen bond, on close approach form hydrogen bond, threethree between GC pairs and between GC pairs and two two between AT- keeps the between AT- keeps the strands in correct complementary orientation.strands in correct complementary orientation.

3) Base stacking3) Base stacking. Stacking interactions are a form of . Stacking interactions are a form of van der waals interaction. Base stacking interactions van der waals interaction. Base stacking interactions are among the aromatic nucleobases. Interaction are among the aromatic nucleobases. Interaction between stacked G and C bases are greater than between stacked G and C bases are greater than those between stacked A and T bases, which largely those between stacked A and T bases, which largely accounts for the greater thermal stability of DNAs with accounts for the greater thermal stability of DNAs with a high G+C contenta high G+C content

4) Electrostatic interaction4) Electrostatic interaction. DNA external surface, . DNA external surface, sugar-phosphate backbone possesses –ve charged sugar-phosphate backbone possesses –ve charged phosphate group. Repulsion between nearby phosphate group. Repulsion between nearby phosphate groups- potentially destabilizing force- phosphate groups- potentially destabilizing force- minimized by shielding effects of divalent cations ie. minimized by shielding effects of divalent cations ie. Mg2+. Mg2+.

The DNA helixThe DNA helixThe geometry of DNAThe geometry of DNA

The biologically most common form of DNA is known as The biologically most common form of DNA is known as B-DNA, - structural features first noted by Watson and B-DNA, - structural features first noted by Watson and Crick together with Rosalind Franklin and other. Crick together with Rosalind Franklin and other.

DNA is flexible molecule. It can assume several distinct DNA is flexible molecule. It can assume several distinct structural depending on its base pair sequence and/or structural depending on its base pair sequence and/or isolation conditions. Each molecular form possesses the isolation conditions. Each molecular form possesses the same no. of base pairs. same no. of base pairs.

DNA can assume different conformations becoz DNA can assume different conformations becoz deoxyribose is flexible and the C1-N- glycosidic linkages deoxyribose is flexible and the C1-N- glycosidic linkages rotates. rotates.

A-DNAA-DNA When DNA become partially dehydrated, it assumes the When DNA become partially dehydrated, it assumes the A A

formform.. The base pairs no longer at right angleThe base pairs no longer at right angle They tilt 20° away from the horizontalThey tilt 20° away from the horizontal Distance between adjacent base pairs slightly reduced (11bp Distance between adjacent base pairs slightly reduced (11bp

helical turn instead or 10.4bp found in B form)helical turn instead or 10.4bp found in B form) Each turn of double helix occur in 2.5nm, instead of 3.4nmEach turn of double helix occur in 2.5nm, instead of 3.4nm Diameter swell to 2.6nm instead of 2.4 nmDiameter swell to 2.6nm instead of 2.4 nm The A form of DNA is observed when it is extracted with The A form of DNA is observed when it is extracted with

solvents such as ethanol.solvents such as ethanol. Significance of A-DNA under cellular conditions- structure of Significance of A-DNA under cellular conditions- structure of

RNA duplexes and RNA/DNA duplexes formed during RNA duplexes and RNA/DNA duplexes formed during transcription. transcription.

Z-DNAZ-DNA

Named for it zigzag conformationNamed for it zigzag conformation Diameter = 1.8nm, slimmer than B-DNA= 2.4 nm.Diameter = 1.8nm, slimmer than B-DNA= 2.4 nm. Twisted into left-handed spiral with 12bp per turn, B Twisted into left-handed spiral with 12bp per turn, B

DNA= 10.4 bpDNA= 10.4 bp Each turn occur in 4.5nm compared with 3.4 nm for B-Each turn occur in 4.5nm compared with 3.4 nm for B-

DNA.DNA. DNA segments with alternating purine-pyrimidines bases DNA segments with alternating purine-pyrimidines bases

(CGCGCG) are most likely to adopt a Z configuration.(CGCGCG) are most likely to adopt a Z configuration. Regions of DNA rich in GC repeats are often regulatory, Regions of DNA rich in GC repeats are often regulatory,

binding specific proteins that initiate/block transcription.binding specific proteins that initiate/block transcription.

Genome structureGenome structure

The genome of each living organism- full inherited The genome of each living organism- full inherited instructions required to sustain living processesinstructions required to sustain living processes

Genome size: the no of base-paired nucleotides, varies Genome size: the no of base-paired nucleotides, varies over an enormous range from less than 1 million bp in over an enormous range from less than 1 million bp in MycoplasmaMycoplasma to greater than 10 to greater than 101010 bp in certain plants. bp in certain plants.

Prokaryotic GenomesProkaryotic Genomes Genome sizeGenome size- The genomes are relatively small The genomes are relatively small - Considerably fewer genes than eukaryotes. Eg: the Considerably fewer genes than eukaryotes. Eg: the E. coliE. coli

chromosome contains about 4.6 Mb that code for 4300 genes. chromosome contains about 4.6 Mb that code for 4300 genes. Coding capacityCoding capacity- Genes are compact and continuous- that is they contain little, Genes are compact and continuous- that is they contain little,

if any, concoding DNA either between/ within gene if any, concoding DNA either between/ within gene sequences. sequences.

Gene expressionGene expression

- The regulation of many functionally related genes is enhanced - The regulation of many functionally related genes is enhanced by organizing them into operons. An operon is a set of linked by organizing them into operons. An operon is a set of linked genes that are regulated as a unit. genes that are regulated as a unit.

Prokaryotes possess additional small pieces of Prokaryotes possess additional small pieces of DNA- DNA- plasmidsplasmids..

Plasmids- have genes that are not present on Plasmids- have genes that are not present on the main chromosome.the main chromosome.

Genes that are not essential for growth and Genes that are not essential for growth and survival but genes that provide growth/survival survival but genes that provide growth/survival advantage: antibiotic resistance genes, unique advantage: antibiotic resistance genes, unique metabolic capacities (N2 fixation, degradation of metabolic capacities (N2 fixation, degradation of aromatic compounds) and virulence (toxins)aromatic compounds) and virulence (toxins)

Eukaryotic genomesEukaryotic genomes Organization of genetic information in eukaryotic Organization of genetic information in eukaryotic

chromosomes- more complex.chromosomes- more complex. Genome sizeGenome size::- Larger than prokaryotes but size does not necessarily a Larger than prokaryotes but size does not necessarily a

measure of the complexity of the organism. Some species measure of the complexity of the organism. Some species accumulated vast amounts of non-coding DNA. accumulated vast amounts of non-coding DNA.

Coding capacityCoding capacity- Although there is enormous coding capacity- majority of DNA Although there is enormous coding capacity- majority of DNA

sequences in eukaryotes do not have coding functions- do not sequences in eukaryotes do not have coding functions- do not possess intact regulatory regions to initiate transcription. possess intact regulatory regions to initiate transcription.

- The function is unknown- some may have regulatory/structural The function is unknown- some may have regulatory/structural roles. Not more than 1.5% of human genome codes for protein. roles. Not more than 1.5% of human genome codes for protein.

Coding continuityCoding continuity- Eukaryotic genes are discontinuous. Non-Eukaryotic genes are discontinuous. Non-

coding sequences (introns) are interspersed coding sequences (introns) are interspersed between sequences called exons.between sequences called exons.

- Exons- code for a gene product.Exons- code for a gene product.- Intron sequences are removed from pre-Intron sequences are removed from pre-

mRNA transcript by splicing mechanism to mRNA transcript by splicing mechanism to produce functional mRNA molecules. produce functional mRNA molecules.

RNARNA Ribonucleic acid is a class of polynucleotides, involved in Ribonucleic acid is a class of polynucleotides, involved in

protein synthesis. protein synthesis. RNA molecules are synthesized in a process referred as RNA molecules are synthesized in a process referred as

transcriptiontranscription.. During transcription- RNA is synthesized thru complementary During transcription- RNA is synthesized thru complementary

base pair formation. base pair formation. The sequence of bases in RNA is therefore specified by the The sequence of bases in RNA is therefore specified by the

base sequence in one of 2 strands in DNA. base sequence in one of 2 strands in DNA. Only 1 DNA strand that acts as Only 1 DNA strand that acts as templatetemplate for synthesis of RNA for synthesis of RNA

molecule- referred as molecule- referred as antisenseantisense (non-coding strand). (non-coding strand). The The nontranscribednontranscribed DNA strand is called DNA strand is called sensesense strand strand

(coding).(coding). The base sequence of the sense strand is the DNA version of The base sequence of the sense strand is the DNA version of

the mRNA used to synthesize the polypeptide product of the mRNA used to synthesize the polypeptide product of gene. gene.

For example, the antisense DNA For example, the antisense DNA sequencesequence

5’- CCGATTACG-3’ is transcribed into the 5’- CCGATTACG-3’ is transcribed into the RNA sequence 3’- GGCUAAUGC-5’. RNA sequence 3’- GGCUAAUGC-5’.

RNA molecules differ from DNA:RNA molecules differ from DNA:

The sugar moiety of RNA is The sugar moiety of RNA is riboseribose. . DNA=deoxyribose. DNA=deoxyribose.

The nitrogenous bases in RNA differ from The nitrogenous bases in RNA differ from those observed in DNA. Instead of those observed in DNA. Instead of thymine, RNA molecules use uracil thymine, RNA molecules use uracil (Adenine base pairing with uracil).(Adenine base pairing with uracil).

In contrast to double helix DNA, RNA In contrast to double helix DNA, RNA exists as a single strand.exists as a single strand.

Secondary structure of RNASecondary structure of RNA RNA exist as single strand.RNA exist as single strand. RNA can coil back on itself and form a unique RNA can coil back on itself and form a unique

secondary structuresecondary structure The shape of these structures determined by The shape of these structures determined by

complementary base pairing by specific RNA complementary base pairing by specific RNA sequence, as well as base stackingsequence, as well as base stacking

The most prominent types of RNA:The most prominent types of RNA: Transfer RNA (Transfer RNA (tRNAtRNA)) Ribosomal RNA (Ribosomal RNA (rRNArRNA)) Messenger RNA (Messenger RNA (mRNAmRNA) )

Differences between DNA & RNADifferences between DNA & RNA

RNARNA DNADNA

Sugar moiety is Sugar moiety is riboseribose Sugar moiety is Sugar moiety is deoxyribosedeoxyribose

Nitrogenous base Nitrogenous base Adenine, Adenine, Urasil, Urasil, Guanine, CytosineGuanine, Cytosine

Nitrogenous baseNitrogenous base

Adenine, Adenine, Thyamine,Thyamine,

Guanine, CytosineGuanine, Cytosine

Exist in Exist in single strandsingle strand Exist in Exist in double helixdouble helix

Content of A and U, as Content of A and U, as well as G and C are well as G and C are equalequal

Content of A and T, as Content of A and T, as well as G and C are well as G and C are equalequal

ExerciseExercise

Consider the following antisense DNA:Consider the following antisense DNA:

5’-CGCTATAGCGTTTCAT-3’5’-CGCTATAGCGTTTCAT-3’- Determine the sequence of its Determine the sequence of its

complementary strandcomplementary strand- Determine the mRNA transcriptDetermine the mRNA transcript- Determine the antisense mRNA Determine the antisense mRNA

sequences. sequences.

QUIZQUIZ

When DNA is heated, it denatures, that is the When DNA is heated, it denatures, that is the strands separate. Determine which of the strands separate. Determine which of the following molecules will denature first as the following molecules will denature first as the temperature is raised. Why?temperature is raised. Why?

a)a) 5’- GCATTTCGGCGCGTTA-3’5’- GCATTTCGGCGCGTTA-3’

3’- CGTAAAGCCGCGCAAT-5’3’- CGTAAAGCCGCGCAAT-5’

b) 5’- ATTGCGCTTATATGCT-3’b) 5’- ATTGCGCTTATATGCT-3’

3’- TAACGCGAATATACGA-5’3’- TAACGCGAATATACGA-5’

QUIZQUIZ

Consider the following sense DNA sequence:Consider the following sense DNA sequence:

5’-GCATTCGAATTGCAGACTCCT-3’5’-GCATTCGAATTGCAGACTCCT-3’

a)a) Determine the sequence of its Determine the sequence of its complementary strandcomplementary strand

b)b) Determine the mRNA and antisense RNA Determine the mRNA and antisense RNA sequencessequences

Transfer RNA (tRNA)Transfer RNA (tRNA)

Transfer RNATransfer RNA tRNA transport amino acids to ribosomes for tRNA transport amino acids to ribosomes for

assembly into protein assembly into protein Comprising about 15% of cellular RNA, Comprising about 15% of cellular RNA, Average length of tRNA = 75 nucleotidesAverage length of tRNA = 75 nucleotides tRNA molecules bound to a specific amino tRNA molecules bound to a specific amino

acid- cells possess at least 1 type of tRNA acid- cells possess at least 1 type of tRNA for each of the 20 amino acids commonly for each of the 20 amino acids commonly found in protein.found in protein.

tRNA- cloverleaf structure.tRNA- cloverleaf structure. The structure allows it to perform 2 The structure allows it to perform 2

important functions:important functions:- The 3’-terminus- forms a covalent bond to a The 3’-terminus- forms a covalent bond to a

specific amino acidspecific amino acid- Anticodon loop- contains 3-base-pair Anticodon loop- contains 3-base-pair

sequence that is complementary to the sequence that is complementary to the DNA triplet code for the specific amino acid.DNA triplet code for the specific amino acid.

tRNA structuretRNA structure

Ribosomal RNARibosomal RNA rRNA is the most abundant RNA in living cellsrRNA is the most abundant RNA in living cells rRNA is the component of ribosomesrRNA is the component of ribosomes Ribosomes = cytoplasmic structures that Ribosomes = cytoplasmic structures that

synthesized proteinssynthesized proteins Ribosomes of prokaryotes and eukaryotes are Ribosomes of prokaryotes and eukaryotes are

similar in shape and function- differ in size and similar in shape and function- differ in size and chemical composition.chemical composition.

Both types of ribosome consist of 2 subunits of Both types of ribosome consist of 2 subunits of unequal size.unequal size.

Prokaryotic ribosome: 50 S and 30 S subunit.Prokaryotic ribosome: 50 S and 30 S subunit. Eukaryotic ribosome: 60 S and 40 S subunit. Eukaryotic ribosome: 60 S and 40 S subunit.

Ribosomal RNARibosomal RNA

Messenger RNAMessenger RNA mRNA is the carrier of genetic information from DNA for mRNA is the carrier of genetic information from DNA for

the synthesis of proteinthe synthesis of protein mRNA is transcribed from a DNA template, and carries mRNA is transcribed from a DNA template, and carries

coding information to the sites of protein synthesis: the coding information to the sites of protein synthesis: the ribosomesribosomes

Prokaryotic mRNA;Prokaryotic mRNA;- polycistronic- contain coding information for several polycistronic- contain coding information for several

polypeptide chainspolypeptide chains- Are translated into proteins by ribosomes Are translated into proteins by ribosomes

during/immediately after they are synthesizedduring/immediately after they are synthesized

Eukaryotic mRNAEukaryotic mRNA:: Typically codes for a single polypeptide-Typically codes for a single polypeptide-

monocistronic.monocistronic. Are modified extensively- capping at the Are modified extensively- capping at the

5’-residue, splicing (removing of introns), 5’-residue, splicing (removing of introns), attachment of poly A tails. attachment of poly A tails.

Ruptured bacterial cells or isolate eukaryotic nucleusRuptured bacterial cells or isolate eukaryotic nucleus

- to expose the nucleic acid- to expose the nucleic acid Bacterial nucleic acid can be precipitated by treating cells Bacterial nucleic acid can be precipitated by treating cells

with alkali and lysozyme (an enzyme that degrades with alkali and lysozyme (an enzyme that degrades bacterial cell walls by breaking glycosidic bonds)bacterial cell walls by breaking glycosidic bonds)

Partially degraded protein is extracted using certain Partially degraded protein is extracted using certain solvents (phenol & chloroform)solvents (phenol & chloroform)

Eukaryotic nuclei can be treated with detergents/ solvents Eukaryotic nuclei can be treated with detergents/ solvents to release their nucleic acid.to release their nucleic acid.

Precipitating the DNA with an alcohol Precipitating the DNA with an alcohol

- usually ice-cold ethanol or isopropanol. Since DNA is - usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, insoluble in these alcohols, it will aggregate together, giving a giving a pelletpellet upon centrifugation. This step also upon centrifugation. This step also removes alcohol-soluble salt removes alcohol-soluble salt

Nucleic acid extraction protocol

Denaturation and renaturation of Denaturation and renaturation of DNADNA

Unique properties of nucleic acids- under certain Unique properties of nucleic acids- under certain conditions DNA duplexes reversibly melt (separate) and conditions DNA duplexes reversibly melt (separate) and reanneal (base pair to form duplex again)reanneal (base pair to form duplex again)

Binding forces that hold the DNA double helix can be Binding forces that hold the DNA double helix can be disrupteddisrupted

This process = denaturation, promoted by :This process = denaturation, promoted by :

- heat (most common denaturing method)- heat (most common denaturing method)- low salt concentrations- low salt concentrations- extremes in pH- extremes in pH

- The temp at which one-half of a DNA sample is denatured - The temp at which one-half of a DNA sample is denatured referred as Tm- varies among DNA molecules according to referred as Tm- varies among DNA molecules according to base composition. base composition. - Renaturation DNA can be prepared by maintain the temp. - Renaturation DNA can be prepared by maintain the temp. ~ 25~ 25ooC below denaturing temp.C below denaturing temp.- requires some time because the strands explore various - requires some time because the strands explore various configurations until they achieve the most stable oneconfigurations until they achieve the most stable one

Nucleic acid methodsNucleic acid methods

Most of technique used in nucleic acid research Most of technique used in nucleic acid research are based on differences in molecular weight or are based on differences in molecular weight or shape, base sequences, or complementary base shape, base sequences, or complementary base pairingpairing

Some of the most useful nucleic acid fractionation Some of the most useful nucleic acid fractionation procedure are:procedure are:

ChromatographyChromatography ElectrophoresisElectrophoresis UltracentrifugationUltracentrifugation

Chromatography Chromatography Many of the chromatographic techniques that are Many of the chromatographic techniques that are

used to separate proteins also apply to nucleic used to separate proteins also apply to nucleic acidsacids

Several types of chromatography: ion-exchange, Several types of chromatography: ion-exchange, gel filtration and affinity. gel filtration and affinity.

ObjectivesObjectives : purify nucleic acid of interest or : purify nucleic acid of interest or isolation of individual nucleic acid sequencesisolation of individual nucleic acid sequences

A type of column chromatography that A type of column chromatography that uses a calcium phosphate gel calleduses a calcium phosphate gel called hydroxyapatitehydroxyapatite been used in nucleic acid been used in nucleic acid researchresearch

Hydroxyapatite bind tightly to double-Hydroxyapatite bind tightly to double-stranded nucleic acid than single-stranded stranded nucleic acid than single-stranded nucleic acid moleculesnucleic acid molecules

So dsDNA can be effectively separate So dsDNA can be effectively separate from ssDNA, RNA or other protein from ssDNA, RNA or other protein contaminants by this methodcontaminants by this method

dsDNA can be rapidly isolated by passing a cell dsDNA can be rapidly isolated by passing a cell lysate through a hydroxyapatite columnlysate through a hydroxyapatite column

wash the column with a low concentration of wash the column with a low concentration of phosphate buffer to release only the ssDNA, RNA phosphate buffer to release only the ssDNA, RNA and proteinand protein

Elute the column with a concentrated phosphate Elute the column with a concentrated phosphate buffer tp collect dsDNAbuffer tp collect dsDNA

hydroxyapatiteRNA + protein dsDNA

Affinity chromatography is used to isolate Affinity chromatography is used to isolate specific nucleic acids. specific nucleic acids.

For example, most eukaryotic messenger RNAs For example, most eukaryotic messenger RNAs (mRNAs) have a poly (A) sequences or cellulose (mRNAs) have a poly (A) sequences or cellulose to which poly (U) is covalently attached. The to which poly (U) is covalently attached. The poly(A) sequences specifically bind to the poly(A) sequences specifically bind to the complementary poly(U) in high salt and low complementary poly(U) in high salt and low temperature and can later be released by temperature and can later be released by altering these condition.altering these condition.

ElectrophoresisElectrophoresis

Gel electrophoresis separate nucleic acids on Gel electrophoresis separate nucleic acids on the basis of molecular weight and 3-D structure the basis of molecular weight and 3-D structure in an electric fieldin an electric field

The technique involves drawing DNA molecules, The technique involves drawing DNA molecules, which have an overall negative charge, through which have an overall negative charge, through a semisolid gel by an electric current toward the a semisolid gel by an electric current toward the positive electrode within an electrophoresis positive electrode within an electrophoresis chamber.chamber.

The used gel is typically composed of a purified The used gel is typically composed of a purified sugar component of agar called agarose. sugar component of agar called agarose.

Electrophoresis Electrophoresis Nucleic acids mixture Nucleic acids mixture

placed in wellplaced in well Nucleic acids are -ve Nucleic acids are -ve

charge (phosphate group)charge (phosphate group) Nucleic acid migrate to Nucleic acid migrate to

anodeanode Rate of migration are Rate of migration are

proportional to molecular proportional to molecular sizesize

In genetic engineering, scientists use the In genetic engineering, scientists use the technique to isolate fragments of DNA technique to isolate fragments of DNA molecules that can then be inserted into molecules that can then be inserted into vectors, multiplied by PCR, or preserved in vectors, multiplied by PCR, or preserved in a gene library. a gene library.

Southern blottingSouthern blotting

The unique properties of nucleic acid: under The unique properties of nucleic acid: under certain conditions DNA duplexes reversibly melt certain conditions DNA duplexes reversibly melt and reanneal. and reanneal.

Enable researcher to detect and analyze Enable researcher to detect and analyze particular DNA sequence- to locate specific particular DNA sequence- to locate specific nucleic acid sequences. nucleic acid sequences.

The basis of detecting specific sequence : The basis of detecting specific sequence : nucleic acids hybridization nucleic acids hybridization

Single-stranded DNA from different sources Single-stranded DNA from different sources hybridize if there is a significant sequence hybridize if there is a significant sequence homology.homology.

Hybridization can be used to locate and/ or Hybridization can be used to locate and/ or identify specific genes or other sequenceidentify specific genes or other sequence

Eg. ssDNA from two diff sources (tumor Eg. ssDNA from two diff sources (tumor cell and normal cell) can be screened for cell and normal cell) can be screened for sequence differencessequence differences

Southern blot technique Southern blot technique

1)1) Probe labellingProbe labelling Sequences with known identities- DNA or Sequences with known identities- DNA or

RNA probe is radioactively/fluorescent RNA probe is radioactively/fluorescent labeled.labeled.

2) restriction fragment preparation2) restriction fragment preparation DNA samples to be tested are treated DNA samples to be tested are treated

with restriction enzymes that cut at with restriction enzymes that cut at specific nucleotides sequences to specific nucleotides sequences to produce a restriction fragmentsproduce a restriction fragments

Southern blot technique Southern blot technique

3) electrophoresis3) electrophoresis The mixture of restriction fragments from each The mixture of restriction fragments from each

sample are separated by electrophoresis sample are separated by electrophoresis according to their sizeaccording to their size

Each sample forms a characteristic patterns of Each sample forms a characteristic patterns of bandband

The gel soaked with 0.5M NaOH to convert The gel soaked with 0.5M NaOH to convert dsDNA to ssDNAdsDNA to ssDNA

Southern blot

technique

3) Blotting3) Blotting The DNA fragments are transferred to The DNA fragments are transferred to

nitrocellulose filter paper by placing them on a wet nitrocellulose filter paper by placing them on a wet sponge in a tray with a high salt buffer sponge in a tray with a high salt buffer (nitrocellulose bind strongly to ssDNA)(nitrocellulose bind strongly to ssDNA)

As buffer is drawn through the gel and filter paper As buffer is drawn through the gel and filter paper by capillary action, the DNA is transferred and by capillary action, the DNA is transferred and become permanently bound to nitrocellulose filterbecome permanently bound to nitrocellulose filter

4) hybridization with radioactive probe4) hybridization with radioactive probe Nitrocellulose filter is exposed to radioactively Nitrocellulose filter is exposed to radioactively

labeled probe, which bind to ssDNA with a labeled probe, which bind to ssDNA with a complementary sequencecomplementary sequence

4) hybridization with radioactive probe4) hybridization with radioactive probe Nitrocellulose filter is exposed to a solution Nitrocellulose filter is exposed to a solution

containing radioactively labeled probe.containing radioactively labeled probe. The probe is ssDNA complementary to DNA The probe is ssDNA complementary to DNA

sequence of interest, and it attaches by base pairing sequence of interest, and it attaches by base pairing to restriction fragment of complementary sequenceto restriction fragment of complementary sequence

Eg: mRNA that codes for B-globin binds specifically Eg: mRNA that codes for B-globin binds specifically to the B-globin gene, even though B-globin mRNA to the B-globin gene, even though B-globin mRNA lacks the intron present in the gene- sufficient base lacks the intron present in the gene- sufficient base pairing.pairing.

5) Autoradiography5) Autoradiography Rinse away unattached probeRinse away unattached probe Autoradiograph showing hybrid DNA Autoradiograph showing hybrid DNA

fragmentfragment

UltracentrifugationUltracentrifugation

Equilibrium density gradient ultracentrifugation in Equilibrium density gradient ultracentrifugation in CsCl is one of the most commonly used DNA CsCl is one of the most commonly used DNA separation procedures. separation procedures.

At high speeds, a linear gradient of CsCl is At high speeds, a linear gradient of CsCl is established.established.

Mixture of DNA, RNA and protein migrating Mixture of DNA, RNA and protein migrating through this gradient separate into discrete through this gradient separate into discrete bands at position where their densities are bands at position where their densities are equal to density of CsCl.equal to density of CsCl.

DNA mol. with high Guanine and Cytosine DNA mol. with high Guanine and Cytosine content are content are more densemore dense than those with a than those with a higher proportion of adenine and thyamine.higher proportion of adenine and thyamine.

The difference helps separate heterogenous The difference helps separate heterogenous mixtures of DNA fragmentsmixtures of DNA fragments

Single stranded DNA denser than the double Single stranded DNA denser than the double stranded DNA, so the two can be separated by stranded DNA, so the two can be separated by equilibrium density gradient ultracentrifugation. equilibrium density gradient ultracentrifugation.

DNA SequencingDNA Sequencing

To determine the DNA nucleotide To determine the DNA nucleotide sequences.sequences.

The classical chain-termination method The classical chain-termination method requires a single-stranded DNA template, requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation.that terminate DNA strand elongation.

The DNA sample is divided into four separate The DNA sample is divided into four separate sequencing reactions, containing all four of the sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. and dTTP) and the DNA polymerase.

To each reaction is added only one of the four To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of extension and resulting in DNA fragments of varying length.varying length.

The newly synthesized and labelled DNA The newly synthesized and labelled DNA fragments are heat denatured, and fragments are heat denatured, and separated by size (with a resolution of just separated by size (with a resolution of just one nucleotide) by gel electrophoresis on one nucleotide) by gel electrophoresis on a denaturing polyacrylamide gel.a denaturing polyacrylamide gel.