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Tools used in biotechnology and genetic engineering - Answer-In Silico analysis, DNA microarray analysis, gel electrophoresis, PCR, and RFLP analysis. PCR - Answer-*Polymerase Chain Reaction* technique for quickly and easily making many copies of even a very small amount of DNA Reverse Trans...

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MCB3020 Exam 3 Study Guide
Questions with Verified Answers
Tools used in biotechnology and genetic engineering - Answer-In Silico analysis, DNA
microarray analysis, gel electrophoresis, PCR, and RFLP analysis.

PCR - Answer-*Polymerase Chain Reaction*
technique for quickly and easily making many copies of even a very small amount of
DNA

Reverse Transcriptase - Answer-enzyme that turns single-stranded RNA into double-
stranded DNA

Complementary DNA (cDNA) - Answer-DNA produced from an RNA template using
reverse transcriptase. often used to clone eukaryotic genes in prokaryotes.

*Basic Process:*
Begin with single stranded RNA and apply short poly-T primer

Add reverse transcriptase and four nucleotides (ACGT)

Add on enzyme called RNaseH to cut up the RNA and regenerate the RNA primers

Add DNA polymerase and DNA ligase to synthesize a new strand

Gel electrophoresis - Answer-separates nucleic acids and proteins on the basis of their
size and electrical charge, using a gel that is a polymer

Cloning vector - Answer-provides a mean for transferring genes of interest to a host
organism during cloning.

Restriction enzymes - Answer-Endonucleases that cut double-stranded DNA at specific
recognition sequences.

Naturally produced by bacteria as a defense against viral infection, commonly used to
create recombinant DNA

Blotting - Answer-Technique that combines electrophoresis and hybridization to detect
specific DNA fragments

Southern = DNA
Western = Protein
Northern = RNA

,Recombinant plasmids - Answer-Restriction enzymes (cuts DNA of interest and
bacterial plasmid at specific restriction sites, leaving fragments with sticky ends)

cloning vectors

DNA ligase (seals strands together by catalyzing phosphodiester bonds btwn single-
strand breaks)

Recombinant DNA - Answer-DNA containing fragments from two or more different
sources

Autonomous replication? - Answer-Being able to replicate independently of the bacterial
chromosome. Plasmids are capable of this.

Gene cloning - Answer-1 Isolate plasmid DNA from bacterial cell
2 Isolate DNA of interest
3 Insert gene of interest into plasmid DNA, which forms recombinant DNA
4 Insert recombinant DNA into bacterium such as Ecoli
5 Culture the bacteria, growing many identical copies

Cloning? - Answer-Cloning is the process by which DNA sequence is isolated and
replicated using a cloning vector.

Cloning vectors used in creating Recombinant DNA. - Answer-*Plasmids*
The most common type of cloning vector
Plasmid- a self replicating piece of extrachromosomal DNA found in prokaryotes
Popular bc they can be easily transferred from one organism to another via conjugation
or transformation
Typically carry inserted sequence in the mid-size range (20,000 base pairs or less).
Ex: pBR322 and pUC19

*Bacteriophages*
A virus that infects bacteria
Carries smaller fragments ~9,000-25,000 base pairs

*Cosmids*
Is a hybrid btwn a plasmid and a phage.
Carries larger fragments of ~30,000-47,000 base pairs

*Artificial Chromosomes*
Synthetic chromosomes that contain fragments of DNA integrated into a hose
chromosome
Categories: PI artificial chromosomes (PACs), bacterial artificial chromosomes (BACs),
and yeast artificial chromosomes (YACs)
Carries larger fragments
YACs carry fragments up to 1million base pairs

, Used in the Human Genome Project

Ti plasmid - Answer-Tumor-inducing plasmid (Ti)
induces the formation of tumors in hundreds of plant species. Scientists found a way to
take out the tumor-inducing genes and other non-essential regions. The plasmid can be
used as a cloning vector

Medical significance of comparative genomics analysis? RFLP? - Answer-Comparative
genomics studies the differences among the genomes of different organisms.

Gives info about what makes a pathogen virulent and gives insights into what kinds of
vaccines or therapies will be useful to fight against a virulent pathogen

RFLP? - Answer-*Restriction Fragment Length Polymorphism*

Tool for genetic fingerprinting, genome mapping, localization of genes, paternity testing,
and microbial classification

Highly conserved and repetitive DNA sequences are present in nearly every genome,
including genomes of humans, gram- and gram+ bacteria

Uses fact that every organism has slightly different restriction sites so on the
electrophoretic gel the patterns of restrictive fragments will vary

Every person has a unique DNA fingerprint (useful for forensic evidence or paternity
testing)

Protoplast fusion? Electroporation? Transformation (plasmids picked up by laboratory-
induced competent bacterial cells) - Answer-*Protoplast fusion*
Scientists use enzymes or other means to remove cell walls of multiple cells, creating
protoplasts

Then put protoplasts together in solution and apply an electric shock (electroporation)
which makes them fuse together to form a somatic hybrid (commonly used with fungi)

*Transformation*
-The direct uptake of genetic material from a bacterium's surroundings
-Very rare in nature, commonly used in lab

Industrial and medical products produced via biotechnology - Answer-insulin
alpha, beta, and gamma interferons
alpha1-antritrypsin (treats emphysema)
blood clotting factors (treats hemophilia)
calcitonin (treats osteomalacia)

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