Title: Techniques of Molecular Biology
1Chapter 20
- Techniques of Molecular Biology
2The methods of molecular biology depend upon and
were developed from an understanding of the
properties of biological macromolecules
themselves.
3Part I NUCLEIC ACID
4NUCLEIC ACIDS
DNA and RNA separation by gel electrophoresis
- Principle Linear DNA molecules migrate through
the gel toward the positive pole with different
rates when subject to an electrical field. - The DNA molecules can be visualized by staining
the gel with fluorescent dyes, such as ethidium.
5(No Transcript)
6NUCLEIC ACIDS
- Two matrices polyacrylamide and agarose.
Plyacrylamide has more resoving power. - Pulsed-field gel electrophoresis for long DNAs
(up to several Mb in length).
7According to RNA it is similar, however RNA
sample should be treated with reagents ,e.g.
glyoxal to prevent the formation of base pairs.
NUCLEIC ACIDS
8Restriction Endonuleases Cleaves DNA Molecules at
Particular Sites
NUCLEIC ACIDS
- Restriction enzymes recognize short target
sequences and cut at a defined position within
those sequences. - They can generate different ends flush ends and
staggered ends. - We use them to break large DNA into manageable
fragments.
9NUCLEIC ACIDS
Recognition sequences and cut sites of various
endonucleases
10- How we name them??
- Take EcoRI for example
- Eco E. coli
- I the first one
11Hybridization probes can identify
electrophoretically separated DNA and RNA
NUCLEIC ACIDS
- Southern blot named after Edward Southern
- DNA fragments, generated by digestion of a DNA
molecule by a restriction enzyme, are run out on
an agarose gel. - Once stained, a pattern of fragments is seen.
- When transferred to a filter and probed with a
DNA fragment homologous to just one sequence in
the digested molecule, a single band is seen,
corresponding to the position on the gel of the
fragment containing that sequence.
12NUCLEIC ACIDS
One example of southern blot
13DNA Cloning
NUCLEIC ACIDS
- Some termsDNA cloning vector insert
DNAlibrary a population of identical vectors
that each contains a different DNA insert.
14NUCLEIC ACIDS
- Characteristics of vector DNAs1.an origin of
replication2.a selectable marker3.sigle sites
for one or more restriction enzymes.
15How to clone DNA in plasmid vectors
NUCLEIC ACIDS
- A fragment of DNA , generated by cleavage with a
certain restriction enzyme, is inserted into the
plasmid vector linearized by the same enzyme. - The recombinant plasmid is introduced int o
bacteria by transformation. - Cells containing the plasmid can be selected by
growth on the antibiotic to which the plasmid
confers resistance.
16Construction of a genomic DNA library
NUCLEIC ACIDS
- Genomic DNA and vector DNA, digested with the
same restriction enzyme, are incubated together
with ligase - The resulting pool or library of hybrid vectors
is then introduced into E. coli, and the cells
are plated onto a filter placed over agar medium. - The filter is removed from the plate and prepared
for hybridization.
17NUCLEIC ACIDS
18Construction of a cDNA library
NUCLEIC ACIDS
- Isolate mRNA
- use reverse transcriptase to synthesize
complementary DNA strand from mRNA, then use DNA
Pol I to synthesize double stranded DNA. Clone
these cDNAs into appropriate vector (usually
plasmid or phage) - Use Oligo dT primer to hybridize to polyA tail of
mRNA. Primer used by reverse transcriptase for
extension. - Reverse transcriptase is a DNA polymerase which
uses RNA as a template to synthesize
complementary DNA. Cloned from RNA viruses.
19We should note that
NUCLEIC ACIDS
- No introns cloned, nor regulatory sequences
- Genes cloned in this method are only those that
were expressed in the particular tissue mRNA was
isolated from.
20NUCLEIC ACIDS
21NUCLEIC ACIDS
- After having constructed a DNA library,
whether genomic or cDNA, we can use probes to
find specific clones we are interested in.
22Site-directed mutagenesis
NUCLEIC ACIDS
- Using site-directed mutagenesis the
information in the genetic material can be
changed. A synthetic DNA fragment is used as a
tool for changing one particular code word in the
DNA molecule. This reprogrammed DNA molecule can
direct the synthesis of a protein with an
exchanged amino acid.
23Polymerase Chain Reaction
NUCLEIC ACIDS
The Royal Swedish Academy of Sciences awards
1993s Nobel Prize in Chemistry to
For more, click http//nobelprize.org
24NUCLEIC ACIDS
- for contributions to the developments of methods
within DNA-based chemistry - for his invention of the polymerase chain
reaction (PCR) method - for his fundamental contributions to the
establishment of oligonucleotide-based,
site-directed mutagenesis and its development for
protein studies
25Lets look into it in more details
NUCLEIC ACIDS
- Denaturation at 94? the double strand melts
open to single stranded DNA, all enzymatic
reactions stop . - Annealing at 54? The more stable bonds last a
little bit longer (primers that fit exactly) and
on that little piece of double stranded DNA
(template and primer), the polymerase can attach
and starts copying the template. - Extension at 72? This is the ideal working
temperature for the polymerase. The bases
(complementary to the template) are coupled to
the primer on the 3' side (the polymerase adds
dNTP's from 5' to 3', reading the template from
3' to 5' side, bases are added complementary to
the template)
26NUCLEIC ACIDS
27How to determine the sequence of bases in a DNA
molecule
NUCLEIC ACIDS
- The most commonly used method of sequencing DNA -
the dideoxy or chain termination method - was
developed by Fred Sanger in 1977 (for which he
won his second Nobel Prize). The key to the
method is the use of modified bases called
dideoxy bases when a piece of DNA is being
replicated and a dideoxy base is incorporated
into the new chain, it stops the replication
reaction.
28The Nobel Prize in Chemistry 1980
NUCLEIC ACIDS
For more, click http//nobelprize.org
29Elements
NUCLEIC ACIDS
- The DNA to be sequenced in single-stranded form
as a template. - The four nucleotides
- The enzyme DNA polymerase and a primer
- A nucleotide analogue that cannot be extended and
thus acts as a chain terminator
30NUCLEIC ACIDS
Dideoxynucleotides used in DNA sequencing
31NUCLEIC ACIDS
Train termination in the presence of
dideoxynucleotides
32Mechanism
NUCLEIC ACIDS
33NUCLEIC ACIDS
34One example of fluorecent chain-terminating
nucleotides
NUCLEIC ACIDS
35Sequencing Whole Genomes
NUCLEIC ACIDS
36NUCLEIC ACIDS
- First, the source clone is fragmented, producing
a random mixture, and a random sub-clone is
selected for sequencing by the Sanger method. - To ensure that that the whole source clone has
been sequenced, this stretch of DNA must be
sequenced numerous times to produce an ordered
overlapping sequence. - Gaps in this process will occur where a sub-clone
is not fully sequenced.
37Contigs
NUCLEIC ACIDS
- Assemble the short sequences from random shotgun
DNAs into larger contiguous sequences.
38NUCLEIC ACIDS
Contigs are linked by sequencing the ends of
large DNA fragments
39Genome-wide analyses
NUCLEIC ACIDS
- Animal genomes contain complex exon-intron
structure, so it is more difficult to find
protein coding genes.
40NUCLEIC ACIDS
- A variety of bioinformatics tools are required to
identify genes and determine the genetic
composition of complex genomes. - A notable limitation of current gene finder
programs is the failure to identify promoters - EST (expressed sequence tag) is simply a short
sequence read from a larger cDNA.
41NUCLEIC ACIDS
Gene finder methods Analysis of proteincoding
regions in Ciona
42Comparative Genome Analysis
NUCLEIC ACIDS
- Permits a direct assessment of changes in gene
structure and sequence arisen during evolution. - Refines the identification of protein-coding
genes within a given genome.
43What we have learned from comparative genome
analysis
NUCLEIC ACIDS
- Synteny conservation in genetic linkage, between
distantly related animals.
44Part II PROTEINS
45Purification of proteins
PROTEINS
- To purify proteins we make use of their inherent
similarities and differences. - Protein similarity is used to purify them away
from the other non-protein contaminants. - Differences are used to purify one protein from
another. Proteins vary from each other in size,
shape, charge, hydrophobicity, solubility, and
biological activity.
46ImmunoAffinity Chromatography
PROTEINS
47Affinity Chromatography
PROTEINS
- column matrix has a ligand that specifically
binds a protein - specialty affinity columns for binding
recombinant proteins with certain "tags"
48Affinity Chromatography
PROTEINS
49Ion Exchange Chromatography
PROTEINS
- proteins have charges due to amino acid side
groups - bind to charged column matrix depending on their
charge at a particular pH - anionic--negatively charged phosphocellulose,
heparin sepharose, S-sepharose - cationic--positively charged DEAE-sepharose,
Q-sepharose - elute bound proteins from column based on charge
and displacement by salt or pH
50Ion Exchange Chromatography
PROTEINS
51Gel filtrationChromatography
PROTEINS
52Separation of proteins on polyacrylamide gels
PROTEINS
53PROTEINS
- Proteins to be isolated should be treated with
sodium dodecyl sulphate (SDS) and a reducing
agent first to eliminate the secondary, tertiary,
and quarternary structure.
54Protein molecules can be directly sequenced.
PROTEINS
- Edman degradation
- Tandem mass spectrometry
55Edman degradation
PROTEINS
- PITC is used to derivitize the free N-terminus
- trifluoroacetic acid causes cleavage of the
N-terminal amino acid from the protein - acid treatment rearranges derivitized aa to
stable PTH amino acid - the PTH amino acid is separated by chromatography
(HPLC) and identified - N-terminus may be subjected to another round of
degradation
56PROTEINS
57Tandem mass spectrometry
PROTEINS
58Proteomics
PROTEINS
- Proteomics is the large-scale study of proteins,
particularly their structures and functions. This
term was coined to make an analogy with genomics.
- The availability of whole genome sequences in
combination with analytic methods for protein
separation and identification has ushered in the
field of proteomics.
59Proteomics is based on three principal methods
PROTEINS
- 2-D gel electrophoresis for protein separation
- Mass spectrometry for the precise determination
of the molecular weight and identity of a protein - Bioinformatics for assigning proteins and
peptides to the predicted products of protein
coding sequences in the genome.
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