Nucleic acid labeling Radioactive deoxynucleoside triphosphate (dNTP); labeled with H 3 (tritium) or P 32.dNTP Purposes: 1. keep tracking small amounts.

Slides:

Advertisements

Similar presentations

ENZYMES THAT MODIFY DNA AND RNA

Advertisements

PRINCIPLES OF CROP PRODUCTION ABT-320 (3 CREDIT HOURS) LECTURE 14 TECHNIQUES FOR GENETIC ENGINEERING, ISOLATION OF TOTAL CELLULAR DNA NUCLEIC ACID HYBRIDIZATION.

Making Nonradioactive Probes: PCR DIG Labelling. Broad Overall Objective Is Myb61 a single or multicopy gene in A. thaliana Is Myb61 a single or multicopy.

Making Nonradioactive Probes: PCR DIG Labeling. Broad and Long Term Objective To determine the copy number of Myb transcription factor genes in the genome.

DNA Sequencing How do you do it?. DNA Sequencing DNA sequencing – used to determine the actual DNA sequence of an organism. Using a computer, one can.

7.1 cont’d: Sanger Sequencing SBI4UP MRS. FRANKLIN.

The polymerase chain reaction (PCR) rapidly

ZmqqRPISg0g&feature=player_detail page The polymerase chain reaction (PCR)

DNA Replication DNA mRNA protein transcription translation replication Before each cell division the DNA must be replicated so each daughter cell can get.

 E. coli was the subject of the study  OriC is the start of replication  Terminus is the end of replication.

1.) DNA Extraction Follow Kit Grind sample Mix with solution and spin Bind, Wash, Elute.

From Haystacks to Needles AP Biology Fall Isolating Genes  Gene library: a collection of bacteria that house different cloned DNA fragments, one.

Gene expression *The transcription involves synthesis of an RNA from the DNA template and an enzyme called RNA polymerase. *In prokaryotes there is a single.

Polymerase Chain Reaction. PCR Repetitive amplification of a piece or region of DNA Numerous uses –Straightforward amplification & cloning of DNA –RT-PCR.

CHMI E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Nucleic acids: - replication.

(1) End labelling The enzyme polynucleotide kinase is used to transfer the terminal phosphate group of a radiolabelled ATP into 5- hydroxyl termini of.

10/20/20151 Week 3, Lecture 2 How are Probes Labelled? with some contributions from Brian Baranick, Rudy Gonzales, Sue Robles, Cang Thai, Jonnie Burton,

Chapter 5: Exploring Genes and Genomes Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition.

Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry Molecular Genetics and Clinical BiochemistryKSU 9 TH WEEK.

Molecular Testing and Clinical Diagnosis

Nucleotides and Nucleic Acids. Cellular Processes DNA RNA (mRNA) Proteins LipidsCarbohydrates replication transcription translation.

Highlights of DNA Technology. Cloning technology has many applications: Many copies of the gene are made Protein products can be produced.

Primer extension * This labelling technique uses random oligonucleotides (usually hexadeoxyribonucleotide molecules- sequences of six deoxynucleotides)

Chapter 10: Genetic Engineering- A Revolution in Molecular Biology.

Other Molecular Techniques. Blotting Probe synthesis Nick Translation –DNA template preparation –Nick template with DNase I –Fill in gaps with.

Molecular Tools. Recombinant DNA Restriction enzymes Vectors Ligase and other enzymes.

Today’s Outline DNA and DNA Replication Review – Leading Strand vs. Lagging Strand – DNA Pol I vs. DNA Pol III Telomeres Molecular Sculpting – DNA Replication.

PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies.

Semiconservative DNA replication Each strand of DNA acts as a template for synthesis of a new strand Daughter DNA contains one parental and one newly synthesized.

DNA Sequencing Hunter Jones, Mitchell Gage. What’s the point? In a process similar to PCR, DNA sequencing uses a mixture of temperature changes, enzymes.

The Polymerase Chain Reaction (PCR)

710.LC GRADUATE MOLECULAR BIOLOGY 10/31/2011. Lecture 4 Competency Test.

Figure S1 RNA-primed DNA synthesis by T7 DNA polymerase. DNA synthesis on an M13 ssDNA template catalyzed by T7 DNA polymerase requires primers annealed.

DNA Isolation. Nucleic Acid Structure & Function DNA & RNA are composed of Nucleotides A nucleotide consists of three covalently-linked parts: –A nitrogen.

Kevin Chen.  A method of amplifying or copying DNA fragments.

Rajan sharma.  Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.  This method was first time developed.

I. PCR- Polymerase Chain Reaction A. A method to amplify a specific piece of DNA. DNA polymerase adds complementary strand DNA heated to separate strands.

Aim: What are some techniques used in DNA engineering?

Figure : The reactions catalysed by the two different kinds of nuclease. (a) An exonuclease, which removes nucleotides from the end of a DNA molecule.

DNA Sequencing BCH 446.

Gel electrophoresis analysis Automated DNA analyzer.

Chapter 4 Recombinant DNA Technology

Enzymes for manipulating DNA

The Replication of DNA

POLYMERASE CHAIN REACTION TECHNIQUES

Molecular Cloning: Polymerase Chain Reaction

Intro to PCR PCR (polymerase chain reaction) was invented by Kary Mullis in Mullis as a chemist working on small nucleotide strands for a biotech.

PCR uses polymerases to copy DNA segments.

GENETIC ENGINEERING College of Science/ biology department

AMPLIFYING AND ANALYZING DNA.

Restriction digestion and Southern blot

DNA sequencing Direct determination of nucleotide sequence

IB Topics3.4 & 7.2- DNA Replication

Screening a Library for Clones Carrying a Gene of Interest

Sequencing and Copying DNA

CHAPTER 20 DNA TECHNOLOGY.

710.LC GRADUATE MOLECULAR BIOLOGY 9/15/2010

A B - deoxynucleotide (dNTP) dideoxynucleotide (ddNTP)

Polymerases Polymerases are the enzymes that synthesize copies of nucleic acids. DNA polymerase I (DNA pol I) Reverse transcriptase (Rtase) the enzyme.

Bioinformatics Lecture By: Ms AQSAD RASHDA

Phenylthiocarbamide (PTC)

PCR uses polymerases to copy DNA segments.

PCR uses polymerases to copy DNA segments.

PCR uses polymerases to copy DNA segments.

Dr. Israa ayoub alwan Lec -12-

PCR uses polymerases to copy DNA segments.

PCR uses polymerases to copy DNA segments.

PCR uses polymerases to copy DNA segments.

Presentation transcript:

Nucleic acid labeling Radioactive deoxynucleoside triphosphate (dNTP); labeled with H 3 (tritium) or P 32.dNTP Purposes: 1. keep tracking small amounts of nucleic acids in cloning experiments. 2. Production of radioactive probes (probe = single pure sequence) to be used in hybridization experiments.

End labeling Dephosphorylation using phosphatase. Polynucleotide kinase transfers a radiolabeled phosphate groups from an ATP source onto 5’ ends. The product is a labeled DNA with low specific activity.

Nick translation DNA polymerase I (DNA pol I) repairs a nick found naturally, or caused by low concentration of DNase I (nuclease). DNA pol I (nuclease, polymerase) catalyses a strand replacement reaction putting radioactive dNTPs. The result is a highly labeled DNA molecule.

Primer extension DNA is denatured by heating. Primer (six nucleotides) annealed to the single stranded DNA (ssDNA). The Klenow of DNA pol I (devoid of nuclease activity) synthesizes a copy of the template strand putting radioactive dNTP. The product is a DNA of very high specific activity.