Molecular Genetics 2nd Half Flashcards
What is the central dogma
It is the main idea of how you go from DNA to Protein
What is transcription
DNA is used as a template for the synthesis of mRNA occurring in the nucleus
What is efficient about converting to mRNA
Numerous copies can be made in a very short period of time
What is translation
The messenger RNA is decoded by transfer RNA by the help of ribosomes and specific amino acids are assembled into polypeptide chains (Cytoplasm)
What are the three stages of both Transcription and Translation
- Initiation
- Elongation
- Termination
Who developed rapid DNA sequencing
Frederick Sanger in 1977
What was the first mapped genetic disease
Huntington’s
What chromosome does huntington’s affect
Chromosome 4
Who created PCR (Polymerase Chain Reaction)
Kary Mullis
What was the human genome project
Develop technology to map and sequence human and other genomes
What was the goal of the private company Celera Genomics
Trying to privatize the human genome
What is the goal of biotechnology tools and techniques
To manipulate DNA
What is recombinant DNA
A DNA fragment composed of sequences from at least 2 different sources
What are Restriction enzymes
Act as molecular scissors, cut DNA at specific base-pair sequences, and every restriction enzyme has a unique recognition site
How big are recognition sites on restriction enzymes
4-8 base pairs
What is a characteristic of recognition sites
They are palindromic
How are blunt cutters different from sticky end cutters
Blunt ends do not want to bind whereas sticky ends are more useful and can easily reform the H-Bonds required between complementary ends
Where do restriction enzymes come from
They come from bacteria which use the enzymes to defend against viruses (bacteriophages)
What prevents the bacterial DNA from being cut as well
Methylation
What is methylation
Methyl groups are added to nucleotides in the recognition site by methylase enzyme
After methylation, what is the restriction enzyme prevented from doing
Cleaving bacterial DNA
In recombinant DNA, why do sticky ends not fully seal the gaps
The phosphodiester bonds of the backbone must still be repaired by DNA ligase
What are plasmids
Small circular pieces of DNA that naturally occur in the bacterial cytoplasm
How bog are plasmids
Range in size from 2000 to 100 000 base pairs long
In what process are plasmids exchanged between bacteria
During conjugation via the sex pilus
What do plasmids often contain
Antibiotic resistance genes
How are plasmids useful to molecular biologists
A gene of interest can be inserted into a plasmid then this recombinant plasmid can be inserted into a bacteria cell which will then produce the protein from this gene
What is Gel electrophoresis
A technique to separate fragments of DNA by size, once the DNA has been digested by restriction enzymes
Why is DNA negatively charged
Phosphate groups present
What electrode will the DNA run towards
The positive electrode as opposites attract
What end are the wells located on
The negative electrode
What is the purpose of the wells
To load the DNA letting it run into the gel
More migration means what
smaller DNA fragments
What is the agarose gel like
Acts like a sieve or a maze
What is usually located in the first well
A molecular weight marker with pre determined benchmark fragments to compare other DNA to
What do same length bands mean when looking at results for forensics
DNA is matching
What is diabetes Mellitus
A disease in which the blood glucose level is too high due to insufficient production or activity of insulin
What is type 1 diabetes
Inability to produce insulin
What is type 2 diabetes
Low insulin or an inability to use insulin
Who isolated insulin
Dr Frederick Banting and Dr Charles Best at U of T
Who founded a method to determine blood sugar by dip test urinalysis
Helen Free
What is a recognition site
A sequence of bases on DNA strands that restriction enzymes bind to
What is a competent cell
A cell that is able to take up foreign DNA from its sorroundings
What is a vector
A DNA molecule that is used as a vehicle to transfer foreign genetic material into a cell
What is a copy number
The number of plasmids of a specific type within a cell
What is a host cell
A cell that has taken up foreign plasmid or virus and has used its machinery to express the foreign DNA
What is a cloned gene
An identical target of an original target gene that can be introduced by adding it to the host cell and having it copied
What is a restriction map
A diagram that shows restriction enzyme recognition sites and the distances, measured in base pairs, between the sites
What is transformation
The successful introduction of DNA from another source
What is hybridization used for
Identify the cells that contain the introduced plasmids with the desired gene
What is a hybridization probe
A fragment of DNA that is used to detect the presence of complementary nucleotide sequences
What did Cohen and Boyer do
Developed the first recombinant plasmid
Describe Cohen and Boyer’s experiment
Restriction enzyme cleaves amphibian DNA on either side of target gene, the same enzyme then cleaves the double helix of bacterial plasmid DNA, the complementary sticky ends form base pairs with each other and then sealed up with DNA Ligase, the result is a bacterial plasmid with an amphibian gene
What are the four steps to cloning a gene into a plasmid
Cutting the gene of interest, ligate gene into plasmid making a recombinant plasmid, transform bacteria with recombinant plasmid, and selection to isolate bacterial cells that actually take up recombinant plasmid DNA
What is PCR used for
A technique used to create many copies of a certain DNA segment or to amplify DNA
What is the machine behind PCR
thermocycler
What principle is PCR based off of
enzymatic replication of DNA
What enzyme is involved with PCR
Taq DNA Polymerase
Where can Taq DNA polymerase be found
Found in thermophilic bacteria, hot springs
What are the primers used in PCR
DNA primers because RNA primers are too easily degraded
What are the three steps of PCR
Denaturation, Annealing, and DNA synthesis
What happens in denaturation
reaction mixture heated to 94 degrees breaking hydrogen bonds and converting to single stranded DNA, these strands now act like a template for the new strands of DNA
What happens in Annealing
The temperature is lowered to 50-65 degrees, the primers bind to their complementary sequences/anneal, primers are single stranded and 20-30 base pairs in length, serve as a starting point for DNA synthesis, they run in opposite directions so there is a forward primer and a reverse primer
What happens in DNA synthesis
temperature is raised to 72 degrees, the bases area added to the 3’ end of the primer by Taw DNA polymerase elongating the DNA in the 5’ to 3’ direction
How many times are the three steps of PCR repeated to obtain a million copies
repeated approximately 20 times, each cycle doubles the amount of target DNA
What are the three post transcriptional modifications
5” Cap, Poly-A Tail at 3’ End, and Removal of Introns/Joining of exons
What does the 5’ Cap do
protects mRNA from digestion from nucleases in the cytoplasm and binds to ribosomes to initiate translation
What does the Poly-A Tail do
Approximately 200-300 adenine nucleotide bases added to 3’ end by poly-A polymerase
What are exons
Expressed regions, code for part of a protein
What is an intron
interspersed among exons, do not code for protein so they must be removed
What removes introns and joins exons
Spliceosomes remove the introns which are degraded in the nucleus and join exons by phosphodiester bonds
Why is proofreading not necessary in mRNA
errors are less detrimental because hundreds of mRNA are made from the same gene, there will be enough correct copies to produce protein required
Describe Transcription in prokaryotes
No nuclear membrane, transcription and translation are coupled and occur together, no introns in genes of prokaryotes
explain the outcome of how a single gene can code for a variety of proteins
human genome consists of 30-35000 genes however the body can produce well over 100 000 different proteins via ALTERNATIVE SPLICING PATTERNS
In Translation, what is the start codon and what are the 3 stop codons
AUG = Start Codon
UAA, UAG, UGA = 3 Stop Codons
