molecular biology techniques – a primer

Molecular Biology Techniques – A Primer

1

The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.

Hybridization---the base-pairing characteristics of DNA and RNA

DNA cloning--- DNA polymerase, restriction endonucleases and DNA ligase

PCR---Thermophilic DNA polymerase

2

Topic 1: Nucleic acidsNucleic acids1. Electrophoresis2. Restriction3. Hybridization4. DNA Cloning and gene expression5. PCR6. Genome sequence and analysis

3

1. Gel electrophoresis separates 1. Gel electrophoresis separates DNA and RNA molecules DNA and RNA molecules according to according to sizesize, , shapeshape and and topological propertiestopological properties

Gel matrix is an inserted, jello-like porous material that support and allows macromolecules to move through. Agarose and polyacrylamide are two different gel matrices

Electrophoresis

4

DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+)

Linear DNA molecules are separated according to size

The mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed

Electrophoresis

5

DNA separation by gel electrophoresis

large moderate small After electr

6

To separate DNA of different size ranges

Narrow size range of DNA: use polyacrylamide

Wide size range of DNA: use agarose gel

Very large DNA(>30-50kb): use pulsed-field gel electrophoresis

Electrophoresis

7

pulsed-field gel electrophoresis

Switching between two orientations: the larger the DNA is, the longer it takes to reorient

Electrophoresis

8

Restriction endonucleases Restriction endonucleases cleave DNA molecules at cleave DNA molecules at particular sitesparticular sites

Nucleic acid Why use endonucleases?

--To make large DNA molecules break into manageable fragments

Restriction digestion

9

Restriction endonucleases: the nucleases that cleave DNA at particular sites by the recognition of specific sequences

The target site recognized by endonucleases is usually palindromic.

e.g. EcoRI5’….GAATTC.….3’ ….CTTAAG….


10

To name a restriction endonuclease:

e.g. EcoRIthe 1st such

enzyme foundEscherichia coli Species category

R13strain


11

Frequency of the occurrence of hexamaeric sequence:

1/4096 (4-6) Randomly


12

Consider a linear DNA molecule with 6 copies of GAATTC:

it will be cut into 7 fragments which could be separated in the gel electrophoresis by size

(The largest fragment) (The smallest fragment)

Digestion of a DNA fragment with endonuclease EcoRI

13

Endonucleases are used to make restriction map: e.g. the combination of EcoRI + HindIII Allows different regions of one molecule

to be isolate and a given molecule to be identified

A given molecule will generate a characteristic series of patterns when digested with a set of different enzymes


14

Different enzymes recognize their specific target sites with different frequency EcoRI Recognize hexameric sequence: 4-6

Sau3A1 Recognize terameric sequence: 4-4

Thus Sau3A1 cuts the same DNA molecule more frequently


15

sticky ends

blunt ends

Recognition sequences and cut sites of various endonucleases


16

The 5’ protruding ends of are said to be “sticky” because they readily anneal through base-pairing to DNA molecules cut with the

same enzyme

Reanneal with its complementary strand or other strands with the same cut


17

DNA hybridization can be DNA hybridization can be used to identify specific used to identify specific DNA moleculesDNA molecules

Nucleic acid Hybridization: the process of

base-pairing between complementary ssDNA or RNA from two different sources

DNA hybridization

18

Probe: a labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence

Labeling of DNA or RNA probes

End labeling: put the labels at the endsUniform labeling: put the labels internally

Radioactive labeling: display and/or magnify the signals by radioactivity Non-radioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding - substrate application (signal release)

End labelingSingle stranded DNA/RNA

5’-end labeling: polynucleotide kinase (PNK)

3’-end labeling: terminal transferase

Labeling at both ends by kinase, then remove one end by restriction digestion

---------------------G---------------------CTTAAp5’

5’pAATTC G

Uniformly labeling of DNA/RNANick translation:

DNase I to introduce random nicks DNA polI to remove dNMPs from 3’ to 5’ and add new dNMP including labeled nucleotide at the 3’ ends.

Hexanucleotide primered labeling:

Denature DNA add random hexanucleotide primers and DNA pol synthesis of new strand incorporating labeled nucleotide.

J1 Characterization of clones

Strand-specific DNA probes: e.g. M13 DNA as templatethe missing strand can be

re- synthesized by incorporating radioactive nulceotides

Strand-specific RNA probes: labeled by transcription


J1-5 Southern and Northern blottingDNA on blot RNA on blot

1.Genomic DNA preparation RNA preparation2.Restriction digestion -3.Denature with alkali - 4. Agarose gel electrophoresis 5. DNA blotting/transfer and fixation RNA6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody)


Southern analysis

27

Southern bolt hybridization

bI1 bI2 bI3 bI4 bI5

Northern analysis COB RNAs in S. cerevisiae

mRNA

Pre-mRNAs

Blot type

Target

Probe Applications

Southern DNA DNA or RNA

mapping genomic clonesestimating gene

numbers

Northern RNA DNA or RNA

RNA sizes, abundance,and expression

Western Protein Antibodies protein size, abundance


30

SequencingSequencing

Nucleic acid

Two ways for sequencing: 1. DNA molecules

(radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately.

2. chain-termination method

31

chain-termination method ddNTPs are chain-terminating nucleotides:

the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end

32

The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule

33

DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP

Tell from the gel the position of each G

Fluorescence automated sequencing system

Slab gel electrophoresis..

Fluorescence automated sequencing system

capillary gel electrophoresis

Computerized visualization from a

single lane of an automated sequencer.

Method uses non-radioactive

fluorescent labelling.

38

DNA sequencing gel

4 systems with dNTP+ ddGTP, dNTP+ ddATP d NTP+ ddCTP, d NTP+ ddTTP separately

“read” the sequencing gel to get the sequence of the DNA

39

The shortgun strategy permits a partial assembly of large genome sequence

If we want to sequence a much larger and more complicate eukaryotic genome using the shortgun strategy. What can we do?

Firstly, libraries in different level should be constructed.

NUCLEIC ACIDS

41

The DNA fragment can be easily extracted and sequenced automatically.

Sophisticated computer programs have been developed to assemble the randomized DNA fragment, forming contigs.

A single contig is about 50,000 to 200,000 bp. It’s useful to analysis fruit fly genome that contains an average of one gene every 10 kb.

If we want to analysis human genome, contigs should be assembled into scaffolds.

42

1-16 the paired-end strategy permits the assembly of large genome sequence

The main limitation to producing large contigs is the occurrence of repetitive sequence. (Why?)

To solve this problem, paired-end sequencing is developed.

The same genomic DNA is also used to produce recombinant libraries composed of large fragments between 3~100 kb.

NUCLEIC ACIDS

43

The end of each clone can be sequenced easily, and these larger clones can firstly assemble together.

44

If a larger scaffold is needed, you should use a cloning vector that can carry large DNA fragment, (at least 100kb). BAC is a good choice.

45

1-17 genome-wide analysis The purpose of this analysis is to

predict the coding sequence and other functional sequence in the genome.

For the genomes of bacteria and simple eukaryotes, finding ORF is very simple and effective.

NUCLEIC ACIDS

46

For animal genomes, a variety of bioinformatics tools are required to identify genes and other functional fragments. But the accuracy is low.

47

The most important method for validating protein coding regions and identify those those missed by current current gene finder program is the use of cDNA sequence data.

The mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are sequenced using shortgun method. These sequence are used to generate EST (expressed sequence tag) database. And these ESTs are aligned onto genomic scaffolds to help us identify genes.

48

Part II proteins

49

2-1 specific proteins can be purified from cell extracts

The purification of individual proteins is critical to understanding their function. (why?)

Although there are thousands of proteins in a single cell, each protein has unique properties that make its purification somewhat different from others.

proteins

50

The purification of a protein is designed to exploit its unique characteristics, such as size, charge, shape, and in many instance, function.

51

2-2 purification of a protein requires a specific assay

To purify a protein requires that you have an assay that is unique to that protein.

In many instance, it’s convenient to use a measure for the function of the protein, or you may use the antibody of the protein.

It is useful to monitor the purification process.

proteins

52

2-4 Proteins can be separated from one another using column chromatography

In this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads.

There are various ways columns can be used to separate proteins according to their characteristics.

proteins

53

Ion exchange chromatography

The proteins are separated according to their surface charge.

The beads are modified with either negative-charged or positive-charged chemical groups.

Proteins bind more strongly requires more salt to be eluted.

55

Gel Filtration Chromatography

This technique separate the proteins on the bases of size and shape.

The beads for it have a variety of different sized pores throughout.

Small proteins can enter all of the pores, and take longer to elute; but large proteins pass quickly.

57

2-5 affinity chromatography can facilitate more rapid protein purification If we firstly know our target protein can

specifically interact with something else, we can bind this “something else” to the column and only our target protein bind to the column.

This method is called affinity chromatography.

proteins

58

Immunoaffinity chromatography

An antibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column.

However, sometimes the binding is too tight to elute our target protein, unless it is denatured. But the denatured protein is useless.

59

Sometimes tags (epitopes) can be added to the N- or C- terminal of the protein, using molecular cloning method.

This procedure allows the modified proteins to be purified using immunoaffinity purification and a heterologous antibody to the tag.

Importantly, the binding affinity can change according to the condition. e.g. the concentration of the Ca2+ in the solution.

60

immunoprecipitation We attach the antibody to the bead,

and use it to precipitate a specific protein from a crude cell extract.

It’s a useful method to detect what proteins or other molecules are associated with the target protein.

61

2-6 separation of proteins on polyacrylamide gels

The native proteins have neither a uniform charge nor a uniform secondary structure.

If we treat the protein with a strong detergent SDS, the higher structure is usually eliminated. And SDS confers the polypeptide chain a uniform negative charge.

proteins

62

And sometimes mercaptoethanol is need to break the disulphide bond.

Thus, the protein molecules can be resolved by electrophoresis in the presence of SDS according to the length of individual polypeptide.

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

63

2-7 antibodies visualize electrophoretically-separated proteins.

The electrophoretically separated proteins are transferred to a filter. And this filter is then incubate in a solution of an antibody to our interested protein. Finally, a chromogenic enzyme is used to visualized the filter-bound antibody

proteins

64

2-8 protein molecules can be directly sequenced Two sequence method: Edman

degradation and Tandem mass spectrometry(MS/MS).

Due to the vast resource of complete or nearly complete genome, the determination of even a small stretch of protein sequence is sufficient to identify the gene.

proteins

65

Edman degradation It’s a chemical reaction in which the

amino acid’s residues are sequentially release for the N-terminus of a polypeptide chain.

66

Step 1: modify the N-terminal amino with PITC, which can only react with the free α-amino group.

Step 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact.

Step 3: identify the released amino acids by High Performance Liquid Chromatography (HPLC).

The whole process can be carried out in an automatic protein sequencer.

68

Tandem mass spectrometry MS is a method in which the mass

of very small samples of a material can be determined.

69

Step 1: digest your target protein into short peptide.

Step 2: subject the mixture of the peptide to MS, and each individual peptide will be separate.

Step 3: capture the individual peptide and fragmented into all the component peptide.

Step 4: determine the mass of each component peptide.

Step 5:Deconvolution of these data and the sequence will be revealed.

71

2-9 proteomics Proteomics is concerned with the

identification of the full set of proteins produced by a cell or a tissue under a particular by a particular set of conditions.

proteins

72

Three principle methods 1. 2-D gel electrophoresis for protein

separation. 2. MS for the precise determination of a

protein. 3. Bioinformatics technology.

73

1-14 shortgun sequencing a bacterial genome

The bacterium H. influenzae was the first free-living organism to have a complete genome sequenced and assembled.

This organism is chosen as its genome is small (1.8 Mb) and compact.

NUCLEIC ACIDS

74

Its whole genome was sheared into many random fragments with an average length of 1kb.

This pieces are cloned into a plasmid vector. And these clones are sequenced respectively.

All these sequence information are loaded into the computer. The powerful program will assemble the random DNA fragment based on containing matching sequence, forming a single continuous assemble, called a contig.

75

To ensure every nucleotide in the genome was captured in the final genome assemble, 30,000 ~ 40,000 clones are needed, which is ten times larger as the genome. This is called 10×sequence coverage.

This method might seem tedious, but it’s much faster and cheaper than the digestion-mapping-sequencing method. As the computer is much faster at assembling sequence than the time required to map the chromosome.

J3 Polymerase chain reaction

J3-1 PCRJ3-2 The PCR cycle J3-3 TemplateJ3-4 PrimersJ3-5 EnzymesJ3-6 PCR optimization

Analysis and uses of cloned DNA

J3-1 PCR The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary to one end of the the DNA target sequence.


J3-2 The PCR cycle• Denaturation: The target DNA

(template) is separated into two stands by heating to 95℃

• Primer annealing: The temperature is reduced to around 55℃ to allow the primers to anneal.

• Polymerization (elongation, extension): The temperature is increased to 72℃ for optimal polymerization step which uses up dNTPs and required Mg2+ .


Template

Primers

Enzymes

Steps of

PCR

J2 nucleic acid sequencing

J3-3 Template•Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR•Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.


J3-4 Primers• PCR primers need to be about 18 to

30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence.

• Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm5oC


Degenerate primers: an oligo pool derived from protein sequence.E.g. His-Phe-Pro-Phe-Met-Lys can generate a primer 5’-CAY TTY CCN TTY ATG AARY= PyrimidineN= any baseR= purine


J3-5 and 6 Enzymes and PCR Optimization• The most common is Taq

polymerase.It has no 3’ to 5’ proofreading exonuclease activity. Accuracy is low, not good for cloning.

• We can change the annealing temperature and the Mg2+ concentration or carry out nested PCR to optimize PCR.


PCR optimization

I.Reverse transcriptase-PCR

II.Nested PCR


Nested PCR

First roundprimers

First roundPCR

Second roundprimers

Second roundPCR

Gene of interest


Reverse transcriptase-PCR

AAA(A)n

5‘-CapmRNA

(dT)12~18 primer anneal5‘-Cap

AAA(A)n

3‘ 5‘

Reverse transcriptasedNTP

5‘-Cap

AAA(A)n

5‘

cDNA:mRNA hybridRegular

PCR

RT-PCR


molecular biology techniques – a primer

Documents

dna separation

dna fragment

dna ligasepcr

characteristics of dna

large dna molecules

thermophilic dna polymerase

molecular weight dna

restriction endonucleases