CHAPTER 20

Techniquesof

Molecular Biology

Nucleic Acids

• Electrophoresis

• PCR

• Sequence

Electrophoresis through a Gel Separates DNA and RNA Molecular

According To Size• Pores in the gel matrix sieve t

he DNA molecules according to this volume.

• The large molecules move slower.

• After a given time, molecule of different size are separated because they have move different distances.

• Circular<linear<supercoiled

Gel matrices

• Polyacrylamide: narrow size range

• Agarose: wide size range

• pulsed-field: very large DNA(>30~50kb)

Restriction Endonuclease Cleave DNA Molecular at Particular Sites

• The large DNA must be broken into manageable fragments for analyzing.

• Restriction Endonuclease can recognize specific short (4~8bp) target sequence.

Enzyme Sequence Cut Frequency

Sau3A1

EcoRI

Not1

5’-GATC-3’

5’-GAATTC-3’

5’-GCGGCCGC-3’

0.25kb

4kb

65kb

Frequency=1/4n ,where n=the number of bps in the recognition sequence

The hydrogen bonds between the 4bps between these cut sites are easily broken to generate 5’protruding ends of 4 nucleotides in length.

DNA Hybridization Can Be Used to Identify Specific DNA Molecules

• Hybridization : the process of base-pairing between complementary single-stranded polynucleotide form two different sources.

• The probe is used to search mixtures of nucleic acids for molecules containing a complementary sequence.

• The probe DNA is labeled , and the mixture being probed is distributed as a library

Two methods for labeling DNA : 1.synthesize new DNA in the presence of a

labeled precursor. 2.add a label to the end of an intact DNA

molecule.

Hybridization Probes Can Identify Electrophoretically-Separated DNAs and RNAs

• DNA fragments, generated by digestion of a DNA molecule by restriction enzyme ,are run out on an agarose gel.

DNA Cloning&

P C R

Isolation of Specific Segment of DNA

• As molecular analysis requires the separation of specific segments of DNA , the ability to purify DNA is very important.

• Recombinant DNA can alter the expression of or generate DNAs.

• The techniques of DNA cloning and ampli-fication by PCR have become essential tools.

DNA Cloning

• The constructing recombinant DNA and maintain them in cells is DNA cloning.

• The vector can provides the information necessary to propagate the cloned DNA.

• The insert DNA includes the target DNA is inserted within the vetor.

Cloning DNA in Plasmid Vectors

• Vector DNAs have 3 characteristics:

• 1.Contain an origin of replication which allow them to replicate in-dependently.

• 2.Contain a selectable mark that allows cells which have the vector to be identified.

• 3.Have single sites for several restriction enzyme.

See the film

Vector DNA Can Be Introduce into Host Organisms by Transformation

• Transformation is the process by which a host organism can take up DNA form its environment.

• Some bacteria have genetic competence to do this, while E.coli must be treated with Ca2+.

Libraries of DNA Molecules Can Be Created by Cloning

• A DNA library is a population of identical vectors that each contains a different DNA insert.

• Genomic libraries are the simplest libraries using insert DNA form total genomic DNA cleaved with a restriction enzyme.

cDNA library• Reverse transcription : a

special DNA polymerase that can make DNA from an RNA template.

• So RNA can be converted into dsDNA copies (cDNAs)

Hybridization Can Be Used to Identify a Specific Clone in a DNA Library • Colony hybridization :a labeled DNA

probe is used to screen a library.

• The same type of positively-charged membrane filter used in the Southern and northern blotting techniques is again used to secure small amounts of DNA for probing.

Chemically Synthesized Oligonucleotides

• Protonate phospho-amidine is the pre-cursor which are chemically protected molecule used in nucleotide addition.

The Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction (PCR) Amplifies DNAs by Repeated Rounds Amplifies DNAs by Repeated Rounds

of DNA replication in Vitroof DNA replication in Vitro

I want to see the movie!

Sequence

Nested Sets of DNA Fragments Reveal Nucleotide Sequences

• Two methods are invented for creating nested sets of DNA molecules:

• 1. They are radioactively labeled at their 5’termini and are then subjected to four different regimens of chemical treatment that cause them to break preferentially at Gs, Cs, Ts, or As.

• 2.Chain termination nucleotides: Sequenators are based.

Dideoxynucleotides used in DNA sequencing&

Chain termination in the presence of dideoxynucleotides

DNA sequencing by the chain termination method

DNA sequencing gel

• When we spike DNA synthesis reactions with ddCTP, ddATP, and ddTTP, then generated four nested sets of fragments.

• These fragments were resolved on a polyacrylamide gel .

Shotgun Sequencing a Bacterial Genome

• The genome was randomly sheared into many random fragments with an average size of 1kb.

• These pieces were cloned into a plasmid recombinant DNA colonies.

• This is called “shotgun” sequencing.• Random recombinant DNA colonies are picked,

processed ,and sequenced.• 10x sequence coverage means every nucleotide in the

genome was sequenced ten times.

The Shotgun Permits a Partial Assembly of Large Genome Sequences

It is necessary to generate a large number of sequencing reads from many short DNA fragment.

• Recombinant DNA, containing a random portion of a human chromosome ,can be isolated from bacterial plasmids and then quickly sequenced using the automated sequencing machines, such as 384-column automated sequencing machine.

The paired-End Strategy Permits the Assembly of Large Genome Scaffolds

The use of BACs (bacterial artificial chromosome) often permits the assignment of multiple contigs into a single scaffold by virtue of sharing several mesgabases.

Genome-Wide Analyses

Gene finder methods: analysis of protein-coding regions in Ciona.

Comparative Genome Analysis

One of the striking findings of Comparative Genome Analysis is the high degree of Synteny , conservation in genetic linkage ,between distantly related animals, such as human and mice, showed in the figure.

Comparison of a 34 kb region of the mouse and human genomes

Example of a BLAST search

PROTEINS

• Purification from cell extract

• Separation through the column chromatography

• Separation on Polyacrylamide gels

• Immunoblotting

• Sequence directly

• proteomics

Specific Proteins Can Be Purified from Cell Extract

• The purification of proteins is a major part of understanding their function.

• Because the studies of the function of a protein in a complex mixture can often lead to ambiguities.

• It is designed to exploit its unique characteristics (size, charge, shape, and function )

Purification of a Protein requires a Specific Assay

• In the immunoblotting, antibody can detect proteins in the similar way of DNA hybridization.

• Incorporation assay: a DNA or RNA polymerase can be assayed by adding the appropriate template and radioactive nucleotide precursor to crude extract.

Preparation of Cell Extracts Containing Active Protein

• Protein is not resilient to temperature, and denatured once released from the cell even in the moderate temperature.

• So most extracts preparation and protein purification is performed at 4℃.

• For this, cells can be lysed by detergent, shearing forces, treatment with low ionic salt, and so on.

Protein Can Be Separated from One Another Using Column Chromatography• Protein fractions are passed though g

lass columns filled with appropriately modified small acrylamide or agarose beads.

• Many ways columns can be used. And each one varies on the basis of different properties of proteins.

• The figure shows separation proteins by their charge or size.

Other chromatography

• Ion exchange chromatography: the proteins are separated by their surface ionic charge. Protein that interact weakly with the beads are release in a low salt buffer. By increasing the concentration of salt, protein with similar charge can be separated.

• Gel filtration chromatography: it does by the basis of size and shape. The beads have a variety of different sized pores throughout.

Affinity Chromatography Can Facilitate More Rapid Protein Purification

• If a protein can binds ATP , it can be applied to a column of beads that are coupled to ATP.

• The protein you need will bind to the column while others will pass through.

• In the immunoaffinity Chromatography, the antibody is just like the ATP.

Separation of Protein on Polyacrylamide Gels

• The SDS coat the proteins and impact on it a uniform negative charge, then the protein behaves as unstructured polymer.

• It can resolve mixtures of proteins according to the length of individual polypeptide chains.

• After electrophoresis, the protein can be visualized with a stain, such as Coomassie brilliant blue.

Wonderful movie

Antibodies Visualize Electrophoretically-Separated Proteins

• An protein is visualized amidst thousands of other protein by the immunoblotting.

• In it, Electrophoretically separated protein are transferred and bound to a filter.

• At last , a chromomeric enzyme is used to visualize the filter bound antibody

Just like it!

Protein Molecule Can Be Directly Sequenced

• Edman degradation : Cyclic degradation of peptides based on the reaction of phenylisothiocyanate (PITC) with the free amino group of the N-terminal residue such that amino acids are removed one at a time and identified as their phenylthiohydantoin derivatives.

Edman degradation

Edman degradation

Proteomics

• Based on 3 principal methods : • Two-dimensional gel electrophoresis for

protein separation.• Mass spectrometry for the precise

determination of molecular weight and identify of a protein .

• Bioinformatics for assigning proteins and peptides to the predicted products of protein-coding sequence in the genome.

Thank you!