Molecular Genetics Flashcards

Question

What happens in the PCR stage of next generation sequencing?

Answer 1

DNA cut into small fragments, physically or using enzymes Fragments denatured using heat, separating strands Short synthetic oligonucleotides are attached to each end of each fragment, and these are attached to a solid support Fragments amplified by PCR. Primers are complementary to the synthetic oligonucleotides

Answer 2

Universal primers, DNA polymerase, and the 4 nucleotides (dNTPs, tagged with fluorescent dyes) are added One nucleotide is added to the new DNA strand in each cycle, and the unincorporated dNTPs are removed Fluorescent colour of the new nucleotide at each location is detected with a camera Fluorescent tag is removed from the nucleotide, and the labelled synthesis cycle repeats

Answer 3

Subset of the genome expressed as RNA in a particular cell or tissue at a particular time Determining which genes are expressed is done by RNA sequencing. DNA copies of RNAs are made first, using reverse transcriptase, then the cDNA fragments are sequenced

Answer 4

PCR Whole genome amplification (WGA)

Answer 5

Molecular cloning

Answer 6

Y= X x 2^n Y is the amplification at cycle number n X is the input target N is cycle number

Answer 7

10 x PCR buffer MgCl2 dNTPs Forward primer Reverse primer DNA or cDNA template DNA polymerase PCR buffers- Tris-KCl Tris-Ammonium sulfate pH 8.3-8.8 (ALKALINE) DNA polymerase- one unit= incorporation of 10nmol dNTPs in 30 min at 72⁰C

Answer 8

Extracted from thermophilic eubacterium: Thermus aquatics strain Introduced into PCR in late 80s Enzyme stable for several hours at 95⁰C

Answer 9

Water baths Automated heated blocks Fast systems Real time detection

Answer 10

Ionic force [Mg+]: high [Mg+] -> low stringency Temperature: high temperature -> high stringency

Answer 11

Gene cloning, discovery and use Monitoring gene expression Mutagenesis Detection of hereditary diseases Detection of microorganisms and viruses Detection and quantification of GMOs Fingerprinting polymorphism, phylogeny Ancient DNA

Answer 12

Consists of amplification of DNA fragments in bacterial hosts using plasmid vectors or phagemid vectors Application: cloning of DNA/genes

Answer 13

Increases the amount of limited DNA samples Useful for forensics and genetic disease research, as well as new technologies such as next generation sequencing, where DNA quantities are limited but many analyses are required Applications: used to amplify traces of DNA ancient DNA

Answer 14

DNA constructed in the laboratory from 2 or more natural or artificial DNA fragments Digestion with the appropriate restriction enzymes Purified fragments are ligated using DNA ligase to produce rDNA rDNA is cloned via insertion into a vector and amplification into a host cell

Answer 15

To clone or make identical copies of genes

Answer 16

Cloned by inserting it into host cells, this is called or transformation, or transfection if host cells are from animals An altered host cell is called transgenic

Answer 17

Biopharmaceuticals- can be used to make human proteins with biomedical applications Gene therapy- attempts to provide a normal copy of the gene to the cells of a patients body Gene analysis- in research labs, biologists use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function

Answer 18

Gene analysis Genome analysis Production of recombinant proteins Production of genetically modified organisms Molecular vaccines

Answer 19

Collections of bacterial clones containing cDNA representing expressed genes in a given issue at specific conditions and corresponding to a specific physiological situation Only expressed genes are represented 1. RNA extraction and mRNA purification 2. cDNA synthesis and production of double stranded DNA 3. Ligation of the adapters and their digestion 4. Ligation of cDNA into the vector 5. Packaging of the rDNA (cDNA in the vector) into the phage capsule 6. Plating the phage library into host bacteria

Answer 20

Collections of bacterial clones containing fragments of genomic DNA digested by a restriction enzyme Entire genome is represented 1. gDNA extraction and purification 2. Digestion of gDNA and vector with a restriction enzyme 3. Ligation of DNA fragments into the digested vector 4. Packaging of the rDNA (gDNA fragments inserted in the vector) into the phage capsule 5. Plating the phage library into host bacteria

Answer 21

Labelled RNA or DNA fragments with a known sequence used to detect a complementary sequence in a DNA or RNA population

Answer 22

Homologous proteins- from the same organisms Heterologous- from different organism

Answer 23

Random sequencing of thousands of clones from cDNA library prepared from a given tissue at specific conditions

Answer 24

Sequencing DNA libraries containing fragments representing the entire genome of a given organism

Answer 25

Physical DNA-base-pair distances from, one gene to another

Answer 26

Order of genes on a chromosome and the relative distances between these genes

Answer 27

Assigning/locating a specific gene to a particular region of a chromosome and determining the location and relative distances between genes on the chromosome

Answer 28

Single gene approach: Northern blotting Quantitative Reverse transcription-Polymerase Chain reaction (qRT-PCR) Global approach: Checking the expression of thousands of genes at the same time -microarray technology -RNA sequencing

Answer 29

Uses antisense RNA to reduce the expression of a gene to check the impact on the physiology Antisense RNA are sequences complementary to target RNA, they hybridise to it and block it's translation They are produced by transforming the target organ usm with a cDNA that is transcribed into antisense RNA OR It uses RNA interference, using small RNA. Small RNA are natural or synthetic double stranded RNA molecules of 20-25 nucleotides complementary to the target RNA They hybridise to the target RNA and block it's translation. They lead to its degradation by specific RNases

Answer 30

Knock out are collections of insertional mutants produced by rDNA technology A vector is used to transfer randomly or specifically a tDNA (transfer DNA) to a location in the genome to alter a gene at that locus Each mutant has an alteration in a single gene By tracing tDNA it is possible to find which gene was affected By identifying the change in the phenotype it is possible to link gene with the function