Nucleic Acids Structures and Techniques PDF

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DR. PEREZ...

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Nucleic Acids Structures and Techniques

Makes use of restriction enzymes( from bacteria) to digest DNA for analysis of fragments generated -provides a framework for locating bases sequences and genes on small genomes or chromosomes Restriction Enzymes

Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites. After a DNA segment has been digested using a restriction enzyme, the resulting fragments can be examined using a laboratory method called gel electrophoresis, which is used to separate pieces of DNA according to their size. -useful in determining inherited genetic defects and obtaining other genetic information and forensics. -let's consider EcoRI, a common restriction enzyme used in labs. EcoRI cuts at the following site:

● A restriction enzyme is a DNA-cutting enzyme that recognizes specific sites in DNA. Many restriction enzymes make staggered cuts at or near their recognition sites, producing ends with a single-stranded overhang. ● If two DNA molecules have matching ends,

When EcoRI recognizes and cuts this site, it always does so in a very specific

they can be joined by the enzyme DNA ligase. pattern that produces ends with DNA ligase seals the gap between the single-stranded DNA “overhangs” molecules, forming a single piece of DNA.Restriction enzymes and DNA ligase are often used to insert genes and other pieces of DNA into plasmids during DNA cloning. ● Enzymes that Hydrolyze nucleic acids are

called nucleases ● Endo: cleaves at an internal location ● Exo: cleaves at the end ● DNase: acts only on DNA ● RNase: acts only on RNA ● Restriction endonuclease: cleave double stranded DNA, isolated chiefly from Bacteria ● There are three types: ○

If another piece of DNA has matching overhangs (for instance, because it has also been cut by EcoRI), the overhangs

Type I: requires ATP, non-specific

can stick together by complementary

cleavage

base pairing. For this reason, enzymes

○ Type 2: no ATP requirement, specific cleavage

that leave single-stranded overhangs are said to produce sticky ends. Sticky ends

○ Type III: requires ATP, specific cleavage are helpful in cloning because they hold ● DNA is a polyanion ( neg charge), so fragments migrates toward the anode( positive

two pieces of DNA together so they can be linked by DNA ligase.

side) ○ Fragments with the smallest kb travel fastest

Not all restriction enzymes produce sticky ends. Some are “blunt cutters,” which cut straight down the middle of a target sequence and leave no overhang. The restriction enzyme SmaI is an example of a blunt cutter:

● ● Restriction endonucleases and gel analysis can be used to generate a physical map if genomes, genes or other segments of DNA

Blunt-ended fragments can be joined to

○ Cut DNA with multiple restrictions enzymes ○ Analyze the restrictions enzymes ○ Analyze fragments on gel

each other by DNA ligase. However, blunt-ended fragments are harder to ligate together (the ligation reaction is less efficient and more likely to fail) because there are no single-stranded overhangs to hold the DNA molecules in position.

○ Map the restriction site ○ Cut DNA with: HinduIII, BamHI and BamHi ■ HindIII cuts once fragment length =4b( 2.8kb+ 1.2kb ○ BamHI: cuts 2.8 kb and 1.2 kb( cuts

-Human chromosomes differ in sequence every 200 to 500 bp creating or eliminating restriction sites

fragments of HindIII) ○ TO FIND FOR THE ENDS!! Look for the similar lines( 1.8 and 0.9kb) ○ HindIII cuts 1.3 fragments

DNA Sequencing: used to determine the primary sequence of nucleic acid

● Chain Terminator ● Automated DNA sequencing ● DNA yields 2 daughters DNA duplexes identical to the parental DNA molecule

Chain Termination Method

● DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule ● DNA is a double helical molecule ● Complementary fashion by DNA polymerase ● Each strand is a template for copying ● DNA polymerase and primer ● Primer: an oligonucleotides that pairs with the end of the template molecule to form dsDNA ● 5’- 3’ direction ● The Sanger technique uses dideoxynucleotides or ddNTPs in addition to typical deoxynucleotides (dNTPs) in the reaction. ddNTPs result in termination of the DNA strand because ddNTPs lack the 3’-OH

Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which copies DNA in vitro. They include: ● A DNA polymerase enzyme ● A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase ● The four DNA nucleotides (dATP, dTTP, dCTP, dGTP) ● The template DNA to be sequenced

However, a Sanger sequencing reaction also contains a unique ingredient: ● Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP

Chain terminator procedure

● Developed by Snagers ○ Fragment of DNA polymerase I ( klenow fragment- polymerase activity) ○ Single strand of DNA and a DNA primer ○ Nucleoside Triphosphate ( dNTPs: dATP, dCTP, Dttp) equal high concentration ○ Small amount of 2’, 3’ ddNTP ○ Primer is complementary to the 3; end of template DNA ○ ddNTP prevents the addition of next nucleotides because 3’ OH needs for phosphodiester bond is replaced with a 3’H

group required for phosphodiester bond formation between nucleotides. Without this bond, the chain of nucleotides being formed is terminated ● Sanger sequencing requires a single-stranded DNA, a DNA primer (either radiolabeled or with a fluorescent tag), DNA polymerase, dNTPs and ddNTPs. Four reactions are set up, one for each nucleotide, G, A, T and C. In each reaction all four dNTPs are included, but only one ddNTP (ddATP, ddCTP, ddGTP or ddTTP) is added. The sequencing reactions are performed and the products denatured and separated by size using polyacrylamide gel electrophoresis. ● Two sets of gels with differents running times can be used to determine the sequence of up to 800 bases of DNA ● Large-scale sequencing is done using automated DNA sequencing ○ Similar set up to chain termination, difference in the type of ddNTPs used

● Automated DNA sequencing utilizes a fluorescent dye to label the nucleotides instead of a radioactive isotope. The fluorescent dye is not an

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Intercalating Agents distort the double helix

environmentally hazardous chemical and has no special handling or disposal requirements. Instead of using X-ray film to read the sequence, a laser is used to stimulate the fluorescent dye. The fluorescent emissions are collected on a charge coupled device that is able to determine the wavelength There is capillary gel and subjected to electrophoresis Detector and laser Forces that stablize the DNA double helix ○ Hydrogen bonds” A:T and G:C( MORE RICH AND STABLE ○ Base stacking: VAN DER WAALS interaction between adjacent base pairs Why is DNA the helical form?think about all neg charged, we will have a lot of repulsion, helical helps to be in a more stable conformation

Ladder form there is a lot spacing 0.6 but in helical is 0.34

Ethidium bromide( dangerous, not used but helpful in the aid of the visualization of DNA separated using gel electrophoresis, acridine orange( is sued in nucleic acid, helpful in determining the cell cycle), actimocylin ( is an antibiotic shown to have anticancer effects. It is shown to have the ability to inhibit transcription by binding DNA at the transcription initiation

complex and preventing elongation

1. DNA Denaturation: double helix collapse into complementary strands 2. We form a random coil that refers to a single strand 3. As we are heating, we compared to native vs denatured 4. In native structure, high absorbance and we are able to detect the difference 5. DNA denaturation results in an increase in absorbance usually 260nm, hyperchromic effect 6. The temperature at which half if the DNA is denatured is defined as the melting temperature

DNA CAN BE RENATURED BY COOLING DOWN THE TEMP

Single stranded DNA can renature to form DNA duplexes. Denatured DNA will denature to reform the duplex structure if the denaturing conditions are removed

G=C the percentage can be low, low melting temperature, the highest GC TEMP, highest melting temp

Renatuaring requires reassociation of the DNA strand nucleation( slowest step, secondary step) and zippering( first order, fast) into a double helix, a process terms reannealing

RECOMBINANT DNA TECHNOLOGY

Clone is a collection of molecules or cells, all identical to an original molecule or cell. To clone a gene is to make many copies such as bacteria

● Genes can be an exact copy of a nature gene ● Gene can be an altered version of a natural gene ● Recombinant DNA technology ● Sickle cells and cyclic fibrous : lack of chloride ion transportation ● Plasmids are very useful in cloning genes: they are no part of DNA, they occur naturally and they are extrachromosomal DNA ● Plasmids are circular dsDNA and they are double stranded ● Plasmids can be cleaved by restriction enzymes, leaving sticky ends ● Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmids ● A cloning vector is the insertion of a foreign DNA into artificial plasmids that serve to carry the foreign DNA insert in E. coli ● Recombinant DNA technology ○ The isolation, amplification and modification of specific DNA sequences and it is also referred to molecular cloning or genetic engineering ● Construction of a recombinant DNA molecule: ○ 1. The cloning vector and the foreign DNA are cut by the same restriction endonuclease ○ 2. The sticky ends of the vector and the foreign DNA fragments anneal and are covalently joined by DNA ligase ○ 3. The results is a chimeric dna containing a portion of the foreign DNA inserted into the vector

● Plasmids are usually present in bacteria, and plasmids can replicate its own DNA independently of the bacteria, which is why it is often used in DNA cloning. Plasmids usually have an antibiotic resistant gene, so the bacteria won't die in the antibiotic. ● CLoaning vectors are plasmids that can be modified to carry new genes ● Plasmids useful as cloning vectors must have ○ A replicator( origin of replication) ○ A selectable marker (antibiotic resistance gene) ○ A cloning site( where insertion of foreign DNA will not disrupt replication or inactivate

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Recombinant DNA

● Pick an appropriate vector and clone foreign DNA ○ Plasmid ○ Bacteriophage ○ Bacteria artificial chromosomes ( BACs) ● Bc sometime sthe size limits the insertion ○ PLASMID: ■ Relatively small ■ Replicate easily ■ Carry resistance genes for antibiotics ■ Contains multiple restriction sites

● 1. Must be able to cut and rejoin DNA a precise locations ○ Must use sequence specific endonucleases called restriction endonucleases on foreign DNA and vector( use of the same restriction enzyme to cut vector and foreign DNA) ● 2. Select a DNA molecule to serve as a carrier( this is known as the vector) ● 3.Prepare and insert foreign DNA ○ Uses DNA ligase to join two pieces of DNA ○ Vector with inserted foreign DNA is known as recombinant DNA ● 4. Introduce vector/insert into host organism ○ This process is known as transformation ○ Screen for host cells replicating the hybrid DNA ● 5. Screen for host cells replicating the hybrid DNA

❖ Plasmids can be used to transform

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Bacteriophages

recipient E coli cells Transformation means the uptake and replication of exogenous DNA by a recipient cell To facilitate transformation, the bacterial cells are rendered somewhat permeable to DNA by CA2+ and a brief 42 degree heat shock The useful upper limit on cloned inserts in plasmids is about 10kbp genes exceed this size So there will be a bacterial cell but its all closed but with the help of ca2+ the cell will have holes and this will allow to take DNA and have heat stock

➔ Clone DNA segments up to 16kb ➔ Produces large amounts DNA in easily purified ➔ 1. Cleavage by restriction enzymes and separate the fragments, remaining DNA contains genes required for infections but is too small to packages ➔ Annela and ligate ➔ 3. Vitro packaging

BAC vectors

★ Clone DNA segments up to 300 kb ★ Allow for cloning of much larger DNA segments

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What if you don't have enough DNA for cloning purposes or southern hybridization?

RecoMBINANT DNA Detects presence of cloned DNA 1. Antibiotic resistance 2. Color change 3. Radioactive...