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Lecture Outlines
Restriction Edonucleases.
- Introduction - Types - Nomenclature - Characteristics - Importance Gel Electrophoresis. DNA Fingerprinting. Recombinant DNA ( DNA cloning) Polymerase Chain Reaction (PCR). - Requirements of PCR - Steps of PCR - Applications of PCR. DNA sequencing.
Nucleases
3
5 Exonuclease
5
3 Exonuclease
Endonuclease
Nomenclature of RE
One of the first type II RE characterized was from the bacterium E. Coli, and was designated EcoR1 . Names reflect origin based on genus and species
EcoR1
E = genus Escherichia co = species coli R = strain RY13 1 = first endonuclease identified
Characteristics of Type II RE
Restriction enzymes recognize and make a cut within
specific palindromic sequences, known as restriction sites, in the DNA. This is usually a 4 or 6 base pair sequence.
CIVIC,
5
3
MADAM
3 5
GAATTC GAATTC
EcoR1
EcoR1 is a restriction enzyme that searches the DNA molecule until it finds the sequence ( - GAATTC - ) of six nitrogen bases.
GAATTC
GAATTC
EcoR1
(Cuts between GA )
AATTC
G
AATTC
HAE III
HaeIII is a restriction enzyme that searches the DNA molecule until it finds the sequence ( -GGCC - ) of four nitrogen bases.
GAGG
CCAG
CTGG
CCTC
-GC-GC-GG-AA-
-GA-
Frequency of cutting
Average distance between cuts is: 4n where n is number of bps in recognition site.
4-base cutter: Cuts DNA into 256 bp average-sized fragments in a random sequence
Gel Electrophoresis
Separates DNA fragments on the basis of charge and size. The larger the fragment, the more difficulty it has moving
through gels. By placing DNA in a gel, then applying a voltage across the gel, the negatively charged DNA will move toward the positive pole. Large fragments lag behind while small fragments move through the gel relatively rapidly.
Isolating genes. Creating new DNA molecules for cloning. DNA fingerprinting.
DNA fingerprinting
Because restriction endonucleases cut specific
sequences they can be used to make DNA fingerprints of different samples of DNA. DNA fingerprinting can help solve crimes, paternity tests, identifying bodies, etc.
DNA fingerprinting
STEPS: 1. Sample DNA cut with restriction enzymes 2. Fragments separated by size using gel electrophoresis 3.The pattern of bands produced is the DNA fingerprint, which is distinguished statistically form other individuals
Recombinant DNA
Recombinant DNA
GAATTC
AATTC
EcoR1
DNA Ligase
G AATTC
Recombinant DNA
G AATTC
AATTC
GAATTC
GAATTC
CTTAAG
EcoRI
G AATTC
G
AATTC
G
CTTAA
CTTAA
AATTC
AATTC
CTTAA
EcoRI sticky end
CTTAA
G
EcoRI sticky end
DNA Ligase
G AATTC CTTAA G
Recombinant DNA
The result is recombinant DNA Recombinant DNA, in which genes from two different
sources - often different species - are combined in vitro into the same molecule.
2.Cut both DNAs with the same restriction enzyme 3. Mix the DNAs; they join by basepairing with DNA ligase Recombinant DNA plasmid
The process of cloning a human gene in a bacterial plasmid can be divided into five steps.
Bacterial clone carrying many copies of the human gene.
4. Put plasmid
into bacterium
For PCR technique, it is essential to have the knowledge of the nucleotide sequence of short segments ( about 20 nucleotides), known as flanking sequences, at each end of target DNA. Steps of PCR 1. Denaturation 2. Renaturation or Annealing 3. Synthesis (chain extension)
Repeated
These three steps are repeated again and again to generate multiples of target DNA.
A large number of free deoxynucleotides (dNTPs) i.e dATP, dCTP, dGTP, and dTTP.
1. Denaturation: On raising the temperature to about 95 C the DNA gets denatured and the two complementary strands separate.
2
2. Renaturation or annealing : As the temperature of the mixutre is slowly cooled to about 550 - 600 C, the primers base pair with the complementary regions flanking target DNA strands. This process is called renaturation or primer annealing.
3. Synthesis (chain extension): In the presence of DNA polymerase (Taq polymerase) the primers are extended by joining the dNTPs complementary to the target DNA strand. The temperature is raised to 720C which is the optimal temperature for Taq polymerase.
3
The above process is repeated. The number of copies doubles in each cycle. 25 to 30 cycles are sufficient for effective DNA amplification.
Applications of PCR
PCR is highly sensitive, it can detect even the presence of single molecule of DNA.
1. PCR in clinical diagnosis Prenatal diagnosis of inherited diseases : PCR is employed in the prenatal diagnosis of inherited diseases by using chorionic villus samples or cells from amniocentesis. Sickle-cell anemia, thalassemia and phenylketonuria can be detected by PCR. Diagnosis of retroviral infections: PCR from is a valuable tool for diagnosis of retroviral infections, e.g., HIV infection.
PCR is used for the detection of bacterial infections e.g., tuberculosis by Mycobacterium tuberculosis.
Diagnosis of cancers: Several virally-induced cancers (e.g., cervical cancer caused by human papilloma virus) can be detected by PCR.
DNA SEQUENCING
DNA SEQUENCING
DNA Sequencing means finding the order of nucleotides
on a single strand of DNA .
Nucleotide order determines Amino acid order, and hence protein structure and function. An alteration in a DNA sequence can lead to an altered or non functional protein leading to harmful effects. Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment.
5' 3'
Normal Linking
1
OH
5'
2-
PO4
Phosphodiester bond
3'
dideoxynuceotide
P 1 R P 2 ddNTP R P R P A R P 2 R P H H R
Can not react
5'
A
Terminated
3'
Sanger's Method:
ATCG TAGCTAGCTA
32P
Keep on going
ATCG TAGCTAGCTA
Template = ddNTPs
32P
A,T,C,G
Analogue
or
ATCGA TAGCTAGCTA
Producing various fragments
STOP
Terminated
Requirements
Multiple copies of template DNA strand. A radioactive tagged primer (a small piece of DNA that
can pair with the template DNA to act as a starting point for replication).
DNA from all four reactions is separated on a gel in side-byside lanes to produce a sequence ladder. Each reaction generates a set of unique fragment lengths ending with a particular dideoxynucleotide.
DNA sequencing technology requires gel electrophoresis system with the ability to separate DNA fragments that separate by one b.p. An advancement of the sangers method is the use of automated sequencing machines using ddNTPs that are each tagged with a different color fluorescent dye.
Instead of performing four different reactions, DNA synthesis occurs in one tube. The sequencing machine then uses a light sensor to read the gel, identifying the bases by their different colors.