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Ahmad Windu Bahari

DNA SQUENCE

In genetics and biochemistry, sequencing means to determine the primary structure


(or primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear
depiction known as a sequence which succinctly summarizes much of the atomic-level
structure of the sequenced molecule. The first DNA sequences were obtained in the early
1970s by academic researchers using laborious methods based on two-dimensional
chromatography. Following the development of dye-based sequencing methods with
automated analysis, DNA sequencing has become easier and orders of magnitude faster.
The term DNA sequencing refers to sequencing methods for determining the order of
the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.
DNA sequencing is the process of determining the nucleotide order of a given DNA
fragment. Thus far, most DNA sequencing has been performed using the chain termination
method developed by Frederick Sanger. This technique uses sequence-specific termination of
a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing
technologies such as Pyrosequencing are gaining an increasing share of the sequencing
market. More genome data is now being produced by pyrosequencing than Sanger DNA
sequencing. Pyrosequencing has enabled rapid genome sequencing. Bacterial genomes can be
sequenced in a single run with several X coverage with this technique. This technique was
also used to sequence the genome of James Watson recently.
The sequence of DNA encodes the necessary information for living things to survive
and reproduce. Determining the sequence is therefore useful in fundamental research into
why and how organisms live, as well as in applied subjects. Because of the key nature of
DNA to living things, knowledge of DNA sequence may come in useful in practically any
biological research. For example, in medicine it can be used to identify, diagnose and
potentially develop treatments for genetic diseases. Similarly, research into pathogens may
lead to treatments for contagious diseases.

Maxam-Gilbert Sequencing
The method requires radioactive labelling at one end and purification of the DNA
fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one
or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series
of labelled fragments is generated, from the radiolabelled end to the first "cut" site in each
molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis
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for size separation. To visualize the fragments, the gel is exposed to X-ray film for
autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA
fragment, from which the sequence may be inferred.

Chain-termination Methods
The classical chain-termination method requires a single-stranded DNA template, a
DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and
modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into
four separate sequencing reactions, containing all four of the standard deoxynucleotides
(dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only
one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-
terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester
bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA
fragments of varying length.

Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS)


The first of the "next-generation" sequencing technologies, MPSS was developed in
the 1990's at Lynx Therapeutics, a company founded in 1992 by Sidney Brenner and Sam
Eletr. MPSS was a bead-based method that used a complex approach of adapter ligation
followed by adapter decoding, reading the sequence in increments of four nucleotides; this
method made it susceptible to sequence-specific bias or loss of specific sequences. Because
the technology was so complex, MPSS was only performed 'in-house' by Lynx Therapeutics
and no machines were sold; when the merger with Solexa later lead to the development of
sequencing-by-synthesis, a more simple approach with numerous advantages, MPSS became
obsolete. However, the essential properties of the MPSS output were typical of later "next-
gen" data types, including hundreds of thousands of short DNA sequences. In the case of
MPSS, these were typically used for sequencing cDNA for measurements of gene expression
levels. Lynx Therapeutics merged with Solexa in 2004, and this company was later purchased
by Illumina.

454 pyrosequencing
A parallelized version of pyrosequencing was developed by 454 Life Sciences. The
method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each
droplet containing a single DNA template attached to a single primer-coated bead that then
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forms a clonal colony. The sequencing machine contains many picolitre-volume wells each
containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate
light for detection of the individual nucleotides added to the nascent DNA, and the combined
data are used to generate sequence read-outs. This technology provides intermediate read
length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD
on the other.

Solexa Sequencing
Solexa has developed a sequencing technology based on reversible dye-terminators.
DNA molecules are first attached to primers on a slide and amplified so that local clonal
colonies are formed (bridge amplification). One type of nucleotide at a time is then added,
and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can
only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled
nucleotides and the dye is chemically removed from the DNA, allowing a next cycle.

SOLiD sequencing
Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool
of all possible oligonucleotides of a fixed length are labeled according to the sequenced
position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase
for matching sequences results in a signal informative of the nucleotide at that position.
Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each
containing only copies of the same DNA molecule, are deposited on a glass slide. Similar to
Solexa sequencing, this technology produces short read lengths at a low price per base.
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PROTEIN SYNTHESIS

Protein synthesis is the process in which cells build proteins. The term is sometimes
used to refer only to protein translation but more often it refers to a multi-step process,
beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA,
which is then used as input to translation.
The cistron DNA is transcribed into a variety of RNA intermediates. The last version
is used as a template in synthesis of a polypeptide chain. Proteins can often be synthesized
directly from genes by translating mRNA. When a protein needs to be available on short
notice or in large quantities, a protein precursor is produced. A proprotein is an inactive
protein containing one or more inhibitory peptides that can be activated when the inhibitory
sequence is removed by proteolysis during posttranslational modification. A preprotein is a
form that contains a signal sequence (an N-terminal signal peptide) that specifies its insertion
into or through membranes; i.e., targets them for secretion. The signal peptide is cleaved off
in the endoplasmic reticulum. Preproproteins have both sequences (inhibitory and signal) still
present.
For synthesis of protein, a succession of tRNA molecules charged with appropriate
amino acids have to be brought together with an mRNA molecule and matched up by base-
pairing through their anti-codons with each of its successive codons. The amino acids then
have to be linked together to extend the growing protein chain, and the tRNAs, relieved of
their burdens, have to be released. This whole complex of processes is carried out by a giant
multimolecular machine, the ribosome, formed of two main chains of RNA, called ribosomal
RNA (rRNA), and more than 50 different proteins. This molecular juggernaut latches onto
the end of an mRNA molecule and then trundles along it, capturing loaded tRNA molecules
and stitching together the amino acids they carry to form a new protein chain.
The genetic code is the set of rules by which information encoded in genetic material
(DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells.
The code defines a mapping between tri-nucleotide sequences, called codons, and amino
acids. With some exceptions, a triplet codon in a nucleic acid sequence specifies a single
amino acid. Because the vast majority of genes are encoded with exactly the same code (see
the RNA codon table), this particular code is often referred to as the canonical or standard
genetic code, or simply the genetic code, though in fact there are many variant codes. For
example, protein synthesis in human mitochondria relies on a genetic code that differs from
the standard genetic code.
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RNA Codon Table


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RECOMBINANT DNA

Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining


two or more sequences that would not normally occur together. In terms of genetic
modification, it is created through the introduction of relevant DNA into an existing
organismal DNA, such as the plasmids of bacteria, to code for or alter different traits for a
specific purpose, such as antibiotic resistance. It differs from genetic recombination in that it
does not occur through natural processes within the cell, but is engineered. A recombinant
protein is a protein that is derived from recombinant DNA.

This is the method of recombinant DNA:


Cloning and relation to plasmids
Plasmids are extrachromosomal
self-replicating circular forms of DNA
present in most bacteria, such as
Escherichia coli (E. Coli), containing
genes related to catabolism and metabolic
activity, and allowing the carrier
bacterium to survive and reproduce in
conditions present within other species
and environments. These genes represent
characteristics of resistance to
bacteriophages and antibiotics and some
heavy metals, but can also be fairly easily removed or separated from the plasmid by
restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of
a selected segment of DNA, which codes for more "reparative" substances, such as peptide
hormone medications including insulin, growth hormone, and oxytocin. In the introduction of
useful genes into the plasmid, the bacteria are then used as a viral vector, which are
encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects,
and increase the amount of cells with the recombinant DNA present within them.
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Chimeric plasmids
When recombinant DNA is then
further altered or changed to host
additional strands of DNA, the molecule
formed is referred to as "chimeric" DNA
molecule, with reference to the
mythological chimera, which consisted as a
composite of several animals. The presence
of chimeric plasmid molecules is
somewhat regular in occurrence, as,
throughout the lifetime of an organism, the propagation by vectors ensures the presence of
hundreds of thousands of organismal and bacterial cells that all contain copies of the original
chimeric DNA.
In the production of chimeric(from chimera) plasmids, the processes involved can be
somewhat uncertain, as the intended outcome of the addition of foreign DNA may not always
be achieved and may result in the formation of unusable plasmids. Initially, the plasmid
structure is linearised to allow the addition by bonding of complementary foreign DNA
strands to single-stranded "overhangs" or "sticky ends" present at the ends of the DNA
molecule from staggered, or "S-shaped" cleavages produced by restriction endonucleases.
Ahmad Windu Bahari

ORIGINAL GEN

Chemical principles govern specific RNA interaction with amino acids. Aptamer
experiments showed that some amino acids have a selective chemical affinity for the base
triplets that code for them. Recent experiments show that of the 8 amino acids tested, 6 show
some RNA triplet-amino acid association. This has been called the stereochemical code. The
stereochemical code could have created an ancient core of assignments. The current complex
translation mechanism involving tRNA and associated enzymes may be a later development,
and that originally, protein sequences were directly templated on base sequences.
Biosynthetic expansion. The standard modern genetic code grew from a simpler
earlier code through a process of "biosynthetic expansion". Here the idea is that primordial
life "discovered" new amino acids (e.g., as by-products of metabolism) and later back-
incorporated some of these into the machinery of genetic coding. Although much
circumstantial evidence has been found to suggest that fewer different amino acids were used
in the past than today, precise and detailed hypotheses about exactly which amino acids
entered the code in exactly what order have proved far more controversial.
Natural selection has led to codon assignments of the genetic code that minimize the
effects of mutations. A recent hypothesis suggests that the triplet code was derived from
codes that used longer than triplet codons. Longer than triplet decoding has higher degree of
codon redundancy and is more error resistant than the triplet decoding. This feature could
allow accurate decoding in the absence of highly complex translational machinery such as the
ribosome.
Information channels: Information-theoretic approaches see the genetic code as an
error-prone information channel. The inherent noise (i.e. errors) in the channel poses the
organism with a fundamental question: how to construct a genetic code that can withstand the
impact of noise while accurately and efficiently translating information? These “rate-
distortion” models suggest that the genetic code originated as a result of the interplay of the
[54]
three conflicting evolutionary forces: the needs for diverse amino-acids , for error-
tolerance and for minimal cost of resources. The code emerges at a coding transition when
the mapping of codons to amino-acids becomes nonrandom. The emergence of the code is
governed by the topology defined by the probable errors and is related to the map coloring
problem.
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WHERE THE CUTTING GEN LOCATION USE H III + ECOR1?

From elektroforesis for the process of restritcion obtained the following


fragment length,
1. NotI: 3018 bp + 806 bp
2. EcoRI: 3018 bp + 806 bp
3. HindIII: 3824 bp
4. SacI: 3711 bp + 113 bp
5. EcoRI dan HindIII: 3018 bp + 762 bp + 44 bp
6. HindIII dan SacI: 3062 bp + 649 bp + 113 bp
7. NotI dan HindIII: 3018 bp + 762 bp + 44 bp
From data we make maping retritcion enzym on plasmid pGEM T Easy like
picture below:

From result of mapping can know that SacI, EcoRI and NotI have two situs
recognition of restriksi where one situs recognition of restriksi ibidem, while HinIII
only owning one recognition situs. Situs recognition of recognition of Ecori and of
Noti have distance of same restriksi.
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HOW TO MAKE RECOMBINANT GEN?

The first step in creating recombinant DNA is to identify and isolate the gene
of interest from the genome. There are immense databases of sequence data from the
human genome project, along with a number of other organisms commonly studied.
Scientists design PCR primers that recognize sequences flanking the gene of interest
that will result in the desired size and include the entire gene and its known regulatory
sequences. After PCR (polymerase chain reaction, there is an accumulation of the
gene as double-stranded DNA. Another important part of the primer design is the
inclusion of endonuclease recognition sites.
The PCR products, essentially the gene of interest, are digested with the
endonuclease(s). The use of two different endonucleases will guarantee the alignment
of the gene in the plasmid by creating two different ends. The plasmid that the gene
will be inserted into is also digested with the same enzymes. A particular plasmid is
chosen to carry the gene of interest for several reasons. Scientists take into account the
size; intended use gene expression, cloning, selection; reporter genes present that can
be used for the selection of bacterial colonies; and the restriction sites available.
If the enzyme leaves a blunt end, then additional steps are carried out to create
sticky ends for ligation into the plasmid. Otherwise, the cut plasmid and gene are
ligated, combined, in a reaction with the enzyme ligase. The mixture is then put into
bacterial cells by various methods and the cells plated.
On the agar plate growing the non-infectious bacterial cells is usually an
antibiotic or reporter substance that the plasmid interacts with to sort out the plasmids
containing the gene of interest and those not containing it. Positive colonies are then
isolated and the experiments proceed depending on the aim. Sometimes it means
isolation of the DNA for placement in a vector for in vitro experiments. Sometimes it
is large scale culture of the bacteria for the production of the recombinant protein.
Recombinant DNA is used to determine the effects of mutations on gene
expression, to determine the effect of the presence of a gene in disease, and to produce
large quantities of proteins needed in medical therapy. It is a tedious process for
scientists, often taking a week for the development of the final plasmid, which doesn't
always work the first time.
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HOW TO EVALUATE RECOMBINANT GEN?

Testing, Evaluation and/or Release - Research plans, protocols and provisions


for containment for recombinant DNA work under field conditions (outside a lab,
growth chamber, containment or cage) require additional information and IBC review.
Environmental safety and risk must be considered for potentially self-replicating
biological material. Investigators should anticipate potential testing, evaluation or
release of recombinant DNA products (at least one year lead time) for preparation and
review of approval documents.
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REFFERENCES

1. Anonymous. 2010. Genetic Code. http://en.wikipedia.org/wiki/Genetic_code


2. Anonymous. 2010. Nucleic acid sequence.
http://en.wikipedia.org/wiki/Nucleic_acid_sequence
3. Anonymous. 2010. Protein biosynthesis.
http://en.wikipedia.org/wiki/Protein_biosynthesis
4. Anonymous. 2010. Requirements and Procedures for Recombinant DNA.
http://biosafety.tamu.edu/institutional-biosafety-committee/ibcreqproc/ibcreqprocdna
5. Prater, A.M. How scientists create recombinant DNA.
http://www.helium.com/items/1295294-how-recombinant-dna-is-created?page=2