Techniques {amplification, DNA} {DNA amplification} can increase number of DNA-fragment copies. In vivo amplification amplifies repeat sequences in fragile X syndrome. In vitro amplification uses cloning or polymerase chain reactions.
purposes
Amplified DNA indicates mutations, translocations, viral and bacterial infections, sex, genealogy, living and extinct species differences, and forensic identification.
DNA synthesis
To make probes or primers, first blocking-group covers 5' or 3' hydroxyl groups of two nucleotides. Then other two hydroxyl-groups react to make phosphodiester bonds. Then acid or base removes blockers. Process repeats to elongate chain.
After restriction-enzyme digestion, DNA fragments can amplify, and relative lengths indicate polymorphisms {Amplified Fragment Length Polymorphism} (AFLP).
After electrophoresis, DNA fragments flow in transparent tubes {cuvette}|, and lasers excite dyes.
Two complementary DNA or RNA single strands can form a double-stranded molecule {hybridization, DNA}|. Two nucleic-acid strands can pair by hydrogen-bonding A and T, or A and U in RNA, and C and G. DNA or DNA and RNA complementary-strand nitrogenous bases can have hydrogen bonding.
arrays
All array spots have hybridization, as cartridge holds array in position. Hybridization measurement depends on dye sensitivity and dynamic range. If array spots are close together, they can cross-hybridize. Evaporation also causes problems, so arrays have humectants, lids, or dewpoint controls. Spotting pins must be 0.2 mm small and clean. Alternatives to spotting include acoustic focusing, multi-nozzle piezoelectric jets, and continuous solid pin spotting. Probes, cDNA arrays, and oligonucleotide arrays are alternative hybridization methods.
Methods {polymerase chain reaction}| (PCR) can make many DNA-sequence copies using heat-stable polymerase, 20-base primers complementary to + strand at one sequence end, and 20-base primers complementary to - strand at other end. Synthesized strands are additional templates, so process doubles copies each primer-annealing, strand-elongation, and dissociation cycle.
purpose
PCR can detect defined sequences in DNA samples. PCR can make stutter bands and add bands resulting from extra nucleotide addition by Taq polymerase.
mRNA amplification
DNA has small amounts, but mRNA has much larger amounts. First, reverse transcription converts mRNA to cDNA and then PCR amplifies cDNA (RT-PCR).
DNA amplification
Machines heat DNA double helix to 94 C for several minutes to make single-stranded DNA. Solution contains DNA polymerase from heat-tolerant organisms and the four bases.
When temperature lowers to 30 C to 65 C for 30 seconds, 20-nucleotide primer DNA binds to DNA, outside region to copy. One primer is for 5' strand, and one primer is for 3' strand. Annealing puts complementary 20-base primer at both ends.
Machines raise temperature to 72 C for some minutes, to allow DNA polymerase and bases to extend both primers beyond other primer region. Now both double-helix molecules have primer on one end and extend beyond other primer on other end. Elongating both strands uses heat-stable DNA polymerase, which synthesizes DNA.
Machines heat DNA to 94 C for several minutes again to extend same primers through other primer at strand ends. Now all synthesized-strand lengths are the same, from one primer through other primer. There are now four DNAs.
Cycles make twice as many DNA strands, and process uses new and old strands again, making chain reactions. Repeating process 30 to 60 times makes millions of copies.
primers
Primers can be genome repetitive sequences, such as Alu repeats. Alu repeats are 300 bases, but smaller region varies little in humans. Alu repeats are in both directions.
primers: nested
After one PCR, second primer that binds inside copied sequence {nested primer} can amplify shorter sequences.
primers: concentration
If one primer has high concentration and one has low, system makes mostly single-stranded DNA {asymmetric PCR}, with no chain reaction.
contamination
Contamination with wrong DNA is common. Negative controls make sure correct DNA amplifies.
Sequence tags {adapter sequence} are in probes.
Single-stranded DNA sequences {oligonucleotide}| {oligo} can have less than 61 bases.
To add deoxyribonucleotides to nucleic acid by DNA polymerase requires oligonucleotides {primer, DNA}|.
Short RNAs or single-stranded DNAs {probe, DNA} {DNA probe} can detect complementary base sequences by hybridization. Probes have 25 to 60 bases and can have 3'-hexyl-amine. Probes attach to last 1500 base pairs closer to transcript 3' ends, where genes have unique short DNA regions. Bacteria and yeast genes have unique primers. Higher organisms have three million different expressed sequence tags (EST).
process
High-concentration purified probes are in 96, 384, or 1536 wells on plastic microtiter plates. Robots take probes from microtiter plate to make same number of spots on glass slides, one slide for each RNA sample to test. Multiple probes test each gene.
Nucleotide sequences {zipcode} can attach to molecules to allow probe complementary nucleotide sequence {zipcode complement} to hybridize.
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Date Modified: 2022.0225