Analysis of nucleic acid

ANALYSIS OF NUCLEIC
ACID EXTRACTS
PRESENT BY:
NAVEED UR REHMAN
BS, MICROBIOLOGY

ANALYSIS OF NUCLEIC ACID EXTRACTS
 CONTENTS
1. NUCLEIC ACID
2. ANALYSIS OF NUCLEIC ACID
3. METHODS OF NUCLEIC ACID
3.1. DNA REASSOCIATION KINETICS
3.2. MICROARRAYS
3.3. CLONING
3

1. NUCLEIC ACID
4

1. NUCLEIC ACID
1.1 Definition
 Nucleic acids are genetic material consist of either one or two long
chains of repeating units called nucleotides
 Consist of a nitrogen base (a purine or pyrimidine) attached to a
sugar phosphate
 Nucleic acids are DNA and RNA
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1. NUCLEIC ACID
1.2 Function
 Nucleic acids are important because they make up genetic
information in living things
 It is passed down from parent to offspring and is found in the
nucleus of the cell
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 2. ANALYSIS OF NUCLEIC ACID
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2. ANALYSIS OF NUCLEIC ACID
 DNA and RNA are nucleic acid molecules that are used to store and
transmit genetic information inside a cell
 Perform analysis on DNA and RNA, it is often necessary to first
extract these molecules from cells or tissues
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3. METHODS OF ANALYSIS OF NUCLEIC
ACID
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3. METHODS OF ANALYSIS OF NUCLEIC ACID
3.1 DNA REASSOCIATION
 DNA is denatured by temperature or by denaturant (e.g. urea)
 Double helix is lost as two strands of DNA held by hydrogen bond
between complementary bases (G:C and A:T) , come apart
 When temperature is lowered or denatured is removed,
complementary strand will reanneal
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3.1 DNA REASSOCIATION
Figure 3.1 Native double stranded DNA is denatured using heat or alkali into single strand. Under
proper condition, single strands will reanneal with strand having complementary sequence
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 When complexity is low, it takes time for all single strands to find
their complement is less
 As complexity is increases, the time it takes for all complementary
strands to reanneal increases
 The genetic complexity or genome size of several soil microbial
communities was assessed using reassociation kinetic
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 By this procedure, they estimate that the community genome size in
undisturbed organic soils was equivalent to 6000-10,000 E. coli
genome
 In comparison, a heavy metal polluted soil contained 350-1500
genome equivalents
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 3.2 Microarrays
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3.2 Microarrays
 It represent an exciting new development in microbial community
 Nucleic acid is the principle on which the technique is based
 Microarrays is the oligonucleotide probes, rather than the extracted
DNA or RNA target
 They are immobilized on a solid surface in a miniaturized matrix
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3.2 Microarrays
 Thousand of probe can be test for hybridization with sample DNA
and RNA
 In contrast to other hybridization technique, the sample nucleic acids
to be probe fluorescently labeled, rather than the probe themselves
 The labeled sample nucleic acids are hybridized to the probes
contain on microarray
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3.2 Microarrays
 Positive signal are detected by using CSLM or other laser
microarray scanning device
Figure 3.2 Microarray scanning device using to detect positive signal
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3.2 Microarrays
3.2.1 Procedure
 Extract mRNA
 Reverse transcribe into cDNA incorporating
fluorescent probe
• Green probe normal / control / wild type sample
• Red is test sample
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Figure 3.2.1 Microarray procedure
used to make hybridized
to microarray

3.2 Microarrays
3.2.1 Procedure
 Hybridize to microarray
 Laser scanner quantitates data
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3.2 Microarrays
3.2.2 Types
 Three basic types of microarray are used in soil ecology i.e.
3.2.2.1 Community genome arrays (CGA)
3.2.2.2 Phylogenic oligonucleotide arrays (POA)
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3.2 Microarrays
3.2.2.1 Community genome arrays (CGA)
 It is used to compare the genome of specific group of organism
 Membrane based reverse sample genome probing
 Different from RSGP in terms of the arraying substrate and signal
detection strategies
 Use nonporous surface for fabrication and fluorescence based
detection
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3.2 Microarrays
 It is used to characterized the relative diversity of organism in a
sample through the use of rRNA sequence based probes
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3.2 Microarrays
Challenges of POA
 Specificity
 Hybridization
 Secondary structure
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3.3 Cloning
 DNA sequence information obtained from environment samples into
two main ways i.e.
1. Cloning DNA extracted from soil directly
2. Cloning of PCR amplified DNA
 In both cases followed by sequencing of cloned DNA
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3.3 Cloning
3.3.1 Direct cloning
 It involves;
 Isolating DNA from soil
 Ligating DNA into vector (most frequently self replicating plasmid)
 Transforming the vector into a competent host bacterium, such as
commercial available E. coli competent cells, where it can be
maintain and multiplied
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3.3 Cloning
 In this way, a recombinant DNA clone library produced
 Once a cloned library is obtained, DNA inserts contained in the
clone can be re-Isolated from the host cells, purified and sequenced
 The clone library can also be screened for biological activity directly
in E. coli
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3.3 Cloning
 This approach circumvent the need to microorganism from
environmental sample
 It also provides a relatively unbiased sampling of the genetic
diversity of sampled environment
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3.3 Cloning
 It has become possible to cloned large fragment (100-300kb) of
DNA into bacterial artificial chromosome (BAC) vectors (Rondon et
al., 2000)
 BAC are low copy number plasmids that can readily maintain large
DNA insert
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3.3 Cloning
 When some of BAC libraries were analyzed, sequence homologous
to the low G + C
 Gram positive Acidobacterium, Cytophagales, and Proteobacteria
were found
 Rondon et al., (2000) also identified clones that expressed lipase,
amylase, nuclease and hemolytic activities
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3.3 Cloning
 Alternative approach for creating large clones libraries from soil
sequences that allows subsequent profiling of microbial
communities is called serial analysis of ribosomal sequence tags
(SARST)
 In this approach, a region of the 16S rRNA gene is amplified by
PCR, such as the IV region (variable in sequence between
taxonomic groups)
30

Analysis of nucleic acid

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