Terms in this set (179)
Adenine and guanine
What forms the DNA backbone
5' to 3' phosphodiester bonds
The DNA double helix has what?
A T G C (hereditary info of the cell) and major / minor grooves
Cysteine and thymine
What are the shapes of mRNA, tRNA, and rRNA?
mRNA - linear
tRNA - clover leaf shaped
rRNA - globular
DNA replication begins at special sites called _______ where in the DNA molecule?
Origins of replication, middle (not the end) where DNA strands will separate to form replication bubbles that expand in both directions, and thousands of these bubbles happen, speeding up the replication of 3 billion base pairs of DNA molecules
*Prokaryotes only have 1 origin of replication
What is assembled during interphase?
Second chromatid containing a copy of DNA is assembled
One strand of the two is old, and one is new
Enzyme that unwinds the DNA, forming a Y shaped replication fork
Attach to each strand of uncoiled DNA to keep them separate
Single stranded binding protein
break and rejoin the double helix, allowing the prevention of knots (if you unwind a twist, the ends will get extra tight and knot up)
DNA polymerase moves from the ______ direction only, synthesizes a new strand that is antiparallel (____)
3' -------> 5'
5' -------> 3'
works continuously as more dna unzips (synthesized 5' -->3')
for the 5-->3 template strand the DNA polymerase has to go back to the replication fork and work away from it. It produces fragments at a time called okazaki fragments vs continuous replication
Connects Okazaki fragments
is an enzyme that creates a small strip of rna primer off of which dna polymerase can work since it can only add to an existing strand
Initiates DNA replication at special nucleotide sequences (called ori of rep) with short segments of RNA nucleotides, called RNA primers
What does DNA replication require?
Once primer is in place, DNA polymerase can succeed in attaching DNA nucleotides to the primer
What replaces the RNA primer?
The RNA strips are later replaced with DNA by DNA polymerase I
DNA Pol I
Replaces base pairs from te primary and does DNA repair
Has a 5' -> 3' exonuclease to take off the primer and can also proof with a 3' to 5' when laying down a new chain
DNA Pol III
Pure replication [euk have different polymerases]
Can do some proofreading and if it makes a mistake, it will go back and use this to replace it
3' to 5' exonuclease
breaks phosphodiester backbone on a single strand of DNA and removes a nucleotide. Exonuclease can only remove from (in this case of 3' end) of the chain
Energy for elongation is provided by ____________________. Breaking the bonds holding the two extra phosphates provides chemical energy for the process (same w/ transcription!). Human rate 50n/s
two additional phophates attached to each new nucleotide
_____ attaches to RNA primers and begins elongation, the adding of DNA nucleotides to the complement strand
What two problems can happen regarding the telomere?
1. Not enough template strand where primase can attach.
2. Last primase is removed => in order to change RNA to DNA, there must be another DNA strand in front of the RNA primer> DNA pol cannot build after removing RNA primer>ultimately that RNA is destroyed by enzymes that degrade RNA left on the DNA, section of the telomere subsequently lost w/ each replication cycle. Prokaryotic DNA is circular so no telomeres (or issue).
enzyme that attaches to the end of template strand and extends the template strand by adding short sequence of DNA over and over (not important code), allowing elongation of lagging strand to continue. However, at the end will still be not enough for primase to attach but this loss of unimportant segment will not cause any problem. Telomerase carries an RNA template: binds to flanking 3' end of telomere that compliments part of its RNA template, synthesizes to fill in over the rest of its template
Do prokaryotics have the issue with telomeres? Why or why not?
No they don't because they have circular DNA, so there's no issue
Where does telomerase attach?
At the flanking region (3' end) of telomere that is complementary to telomerase RNA
One gene one polypeptide
a gene as the DNA segment that codes for a particular polypeptide. Also, genetic code is universal for nearly all organisms and most AAs have more than one codon specifying them (redundancy/degeneracy)
What did the one gene one polypeptide used to be?
Used to be called one gene one enzyme, but was changed because many genes code for polypeptides that aren't enzymes (such as structural proteins or individual components of enzymes)
What does it mean when the genetic code is redundant?
Meaning that certain amino acids are specified by more than one codon
Single stranded template. A triplet of three adjacent nucleotides on the mRNA (codon) codes for one specific amino acid. Since there are 64 possible ways (4x4x4) ways that four nucleotides can be arranged in triplet combinations, there are 64 possible codons. 3 of them are stop codons. Therefore, only 61 codes for amino acids.
Copies the information stored in the strand of DNA
How many possible codons are there? How many of those are stop codons? Therefore, how many code for amino acids?
64 possible, 3 stop codons, and 31 code for amino acids
What is the consensus sequence
Most common sequence of nucleotides at promoter region (the most frequent residues).
Anything that isn't this / variations will cause less tight RNA pol binding and a lower transcription rate
Size of DNA relative to RNA?
RNA is shorter
Short RNA molecule (80 nucleotides) that is used for transporting amino acids into their proper place on the mRNA template
C-C-A-3' end of tRNA attaches to amino acid, and the other portion is the anticodon which bp with the codon in mRNA. Wobbles: exact bp of the 3rd nucleotide in the anticodon and the 3rd nucleotide in the codon is often not required, allowing 45 different tRNA's base-pair with 61 codons that code for amino acid. Transports AA to its mRNA codon.
Shuttles amino acids to the ribosomes and is responsible for bringing the appropriate amino acids into place at the appropriate time. Does this by reading the message carried by the mRNA
Transfer RNAs are the go-betweens of protein synthesis. Each tRNA becomes _____ and attaches to a(n) ________ in the cell's _____ and shuttles it to the ribosome. The charging enzymes involved in forming the bond between the ____ and tRNA require _____
charged, amino acid, cytoplasm, amino acid, energy
nucleolus is an assemblage of DNA actively being transcribed into rRNA. As ribosome, has three binding sites: one for mRNA, one for tRNA that carries a growing polypeptide chain (P site); one for 2nd tRNA that delivers the next aa (A site). Terminations sequences include UAA, UGA, UAG. Together w/ proteins, rRNA forms ribosomes. Ribosome is assembled in nucleolus but large and small subunits exported separately to cytoplasm.
Produced in the nucleolus, makes up part of the ribosomes (which are the sites of protein synthesis)
Provides the template used for sequencing amino acid
Messenger RNA (mRNA)
What is the function of the large and small subunits?
The two subunits form a ribosome that coordinates the activities of the mRNA and tRNA during translation
Three binding sites of ribosomes?
One for the mRNA, one for the tRNA (that carries a growing polypeptide chain "P site") and one for the second tRNA that delivers the next amino acid that will be inserted ("A site", for amino acid)
Which end of tRNA attaches to an amino acid?
The C-C-A- 3' end does
What base pairs with the codon of mRNA?
The anticodon of the tRNA
Is exact base pairing required between the third nucleotide of the tRNA anticodon and the third nucleotide of the mRNA codon?
No, this wobble allows the anticodon of some tRNA's to base pair with more than one kind of codon and as a result, about 45 different tRNA's base pair with the 61 codons that code for amino acids
Building blocks of ribosomes
Three basic steps of protein synthesis?
Transcription, RNA processing, and translation
In transcription, RNA molecules are created by using the DNA molecular as a template. After transcription, RNA processing modifies the RNA molecule with deletions and additions. In translation, the processed RNA molecules are used to assemble amino acids into a polypeptide
Transcription - three stages?
creation of RNA molecules from DNA template. Prokaryotes polycistronic, eukaryotes monocistronic
Initiation, elongation, and termination
Initiation (of transcription)
RNA pol attaches to promoter region on DNA and unzip the DNA into two strands. A promoter region for mRNA transcription often contains the sequence TATA (TATA Box). Most common sequence of nucleotides at promoter region is called the consensus sequence; variations from it cause less tight RNA pol binding à lower transcription rate
Sense vs antisense strand
The sense strand of DNA is the + sense / coding strand, is the same sequence as mRNA (except that thymine is there instead of uracil)
The antisense is the - template strand
Transcription begins at special sequences called ____?
RNA polymerase function
Brings free floating RNA nucleotides to the DNA strand, and also unzips the DNA strand
Elongation (of transcription)
RNA pol unzips DNA and assembles RNA nucleotides using one strand of DNA as template; only one strand is transcribed (from the template/(-) antisense DNA strand; other strand is coding/(+) sense strand for protection against degradation).
Termination (of transcription)
when RNA pol reaches a special sequences often AAAAAAA in eukaryotes.
Note: transcription is occurring in the 3' to 5' direction of the DNA template strand (but synthesis of the RNA strand is, as always, 5' to 3')
Then it will separate from the DNA template, completing the process of transcription. A new RNA has now transcribed, or "copied" the sequence of nucleotide bases directly from the exposed DNA strand
In transcription, elongation of the RNA molecule occurs in what direction?
5' to 3' direction
This is the stage when mRNA is ready to move out of the nucleus, but before it can, the mRNA must be processed aka must be modified.
This newly made mRNA molecule has more nucleotides than it needs to code for protein and it consists of both coding regions and noncoding regions.
What is the promoter region sequence and termination sequence in transcription
Term is AAAAAAA.
Do prokaryotes have introns?
What are the modifications that pre-mRNA goes through? After all of this, what is produced?
1. 5' Cap
2. Poly A tail
3. RNA splicing
4. Alternative splicing
It produces a final mRNA that is shorter than the transcribed mRNA
Exons vs introns
Exons are the regions that express the code for polypeptide and introns are the noncoding regions
the sequence is added to the 5' end of the mRNA; Guanine with 2 phosphate groups => GTP; providing stability for mRNA and point of attachment for ribosomes.
For RNA processing, what provides stability and controls movement of mRNA across the nuclear? What provides stability for mRNA and point of attachment of ribosomes?
Poly A tail
Poly A tail
sequence is attached to the 3' end of the mRNA. Tail consists of 200A; provide stability and control movement of mRNA across the nuclear envelope. (in prokaryotes, polyA tail facilitates degradation!)
Why can prokaryotes begin translation immediately / simultaneously?
prokaryotes generally have ready to go mRNA upon transcription. It is only in eukaryotes that you need the above processing. Because prokaryotes don't need to process their mRNA first, translation can begin immediately/simultaneously. In both prokaryotes and eukaryotes, multiple RNA polymerases can transcribe the same template simultaneously.
Translation. Three stages?
assembly of amino acids based on reading of new RNA; uses GTP as energy source. Three stages are initiation, elongation, and termination
This occurs after RNA processing, when the mRNA leaves the nucleus and searches for a ribosome
in cytoplasm, amino acid attaches to tRNA at 3' end, require 1 ATP --> AMP per AA
The amino-acid tRNA bond that results is a high energy bond, creating an activated amino acid tRNA complex
The mRNA carries message from DNA in the form of?
Codons, a group of three bases or "letters" that corresponds to one of 20 amino acids
Ribosomes contain three binding sites. They are?
A site, P site, and E site
Initiation of translation
small ribosome unit attaches to 5' end of mRNA; tRNA-methionine (with the sequence UAC) attaches to start sequence of mRNA AUG, and large ribosomal unit attaches to form a complete complex. (requires 1 GTP)
Where does the large ribosomal unit bind? Small?
On the P site of ribosome - attaches to the mRNA, forming a complete ribosome with the tRNA
The small binds to a special region near the 5' end of the mRNA
In all organisms the codon for initiation of protein synthesis is ____ which codes for amino acid _____. The tRNA with the complementary anticodon _____ acts as personal shuttle for methionine
AUG, methionine, UAC
What is the process of translocation in translation?
When tRNA binds to the A site (start of elongation) and then the tRNA moves from the A site to the P site, and the A site becomes exposed and a new codon is exposed
Elongation of translation
Addition of amino acids - mRNA contains many codons and as each amino acid is brought to the mRNA, it is linked to its neighboring amino acid by a peptide bond. When many amino acids link up, a polypeptide is formed
next tRNA binds to A site, peptide bond formation, tRNA without methionine is released, the tRNA currently in A site moves to P site (translocation) and the next tRNA comes into A site and repeat process. (req. 2 GTP per link)
Termination of translation
encounters the stop codon UAG, UAA, UGA. Polypeptide and the two ribosomal subunits all release once release factor breaks down the bond between tRNA and final AA of the popypeptide. While polypeptide is being translated, AA sequences is determining folding conformation; folding process assisted by chaperone proteins (requires 1 GTP)
What are the three stop codons?
What happens once the polypeptide is completed?
Interactions among the amino acids give it is secondary and tertiary structures and subsequent processing by the ER or Golgi make final modifications before the protein functions as a structural element or as an enzyme
Help fold proteins - help fold it properly and make the process more efficient
Translation begins on a free floating ribosome; signal peptide at the beginning of the translated polypeptide may direct the ribosome to attach to the ER, in which case the polypeptide is injected into the ER lumen. If injected, polypeptide may be secreted from the cell via Golgi. In general, post-translational modifications (addition of sugars, lipids, phosphate groups to the AAs) may occur. May be subsequently processed by Golgi before it is functional.
Amino acids are placed starting from the 5' end of the mRNA and move all the way down to the 3' end. tRNA codons for matching are 3' to 5'.
Can occur simultaneously with transcription in prokaryotes, but not in eukaryotes. Multiple ribosomes may simultaneously translate 1 mRNA.
What is the start codon in bacteria?
What are the two major types of mutations?
Base substitutions and gene rearrangements
new codon still codes for the same amino acid - those that cause no detectable change in the corresponding gene sequence
new codon codes for a stop codon - early termination of protein synthesis
new codon codes for new amino acid => minor or fatal results as in sickle cell (val replaces glu)
no change in protein fxn
Deletions, duplications, inversions, and translocations and are often caused by chemical and radioactive agents
Result in the loss of DNA or gene, can involve either the loss of a single base or loss of larger portion of DNA. Insertions can have devastating consequences to the gene because translation of the gene causes a frameshift
Can result when changes occur in the orientation of chromosomal regions. Cause harmful effects if the inversion involves a gene or an important sequence involved in the regulation of gene expression
Can result in an extra copy of genes, and are usually caused by unequal crossing over during meiosis or chromosome rearrangements
Occur when portion of 2 different chromosomes (or single chromosome in two different places) breaks and rejoins in a way that the DNA sequence is lost, repeated, or interrupted
when DNA polymerase checks the base pairs of newly attached bases to the growing strand
Checks to makes sure that each newly added nucleotide correctly base pairs with the template strand. If it does not, then the nucleotide is remove and replaced with the correct nucleotide
Enzymes repair errors that escape the proofreading ability of DNA polymerase
Enzymes remove nucleotides damaged by mutagens
The enzymes identify which of the 2 strands of the DNA contain a damage nucleotide and use the complementary strand as a template to repair the error
Repair mechanisms? (3)
2. Mismatch repair
3. Excision repair
DNA is coiled around bundles of 8/9 histones proteins (beads on a string).
Euchromatin vs heterochromatin
Eu - loosely bound to nucleosomes, actively being transcribed
Heterochromatin - tightly packed nucleosomes where DNA is inactive (condense -> darker) and contains a lot of satellite DNA (large tandem repeats of noncoding DNA)
DNA segments that can move to new location on same/different chromosome; 2 types: insertion sequences consist of only one gene that codes for enzyme that just transports it (transposase); complex transposons code for extra: replication, antibiotic resistance, etc. Insertion of transposons into another region could cause mutation (little to no effect).
What kind of effect do transposons have?
The effect of a mutation - may change the expression of a gene, turn on / turn off its expression, or have no effect at all
a nucleic acid (RNA/DNA may be double/single stranded), capsid (protein coat that encloses the nucleic acid; capsomeres assembles to form the capsid), and envelope (surrounds capsid of some viruses; it incorporates phospholipid/protein obtained from cell membrane of host).
Note: bacteriophage = virus that only attacks bacteria. Usually specific to a type of cell (bind to specific receptors) and species. Host range is range of organisms virus can attack
Structure of a virus?
1. Nucleic acid
What kind of nucleic acid does a virus have?
Can be double or single stranded DNA or RNA
What forms the capsid? What exactly is a capsid?
A capsid is a protein coat that surrounds the nucleic acid of a virus and it is made from capsomeres
virus penetrates cell membrane of host and uses host machinery to produce nucleic acids and viral proteins that are then assembled to make new viruses - these viruses burst out of the cell and infect other cells
Compare DNA virus, RNA virus, and retrovirus
- DNA virus: DNA is replicated and form new viral DNA => transcribed to produce viral proteins (DNA + viral proteins assemble to form new viruses).
- RNA virus: RNA serves as mRNA => translated into protein (protein + RNA => new virus).
- Retroviruses: e.g. HIV, ssRNA viruses that use reverse transcriptase to make DNA complement of their RNA => which can go to manufacture mRNA or go into lysogenic cycle (becoming incorporated into DNA host).
What leads to variation in the lytic cycle?
If the nucleic acid is SS or DS, and if it's DNA or RNA
e.g. HIV, ssRNA viruses that use reverse transcriptase to make DNA complement of their RNA => which can go to manufacture mRNA or go into lysogenic cycle (becoming incorporated into DNA host).
What is the fate of DNA complement that retroviruses make?
They can either make mRNA, or they can go into the lysogenic cycle
What is techoic acid?
Teichoic Acids on cell wall of bacterium are used as recognition + binding sites by bacterial viruses that cause infxns; also provide cell wall rigidity: only found on gram-positive bacteria!
What's a prion?
Not viruses or cells. Misfolded versions of proteins in brain that cause normal version to misfold too. Fatal.
What are bacteria?
are prokaryotes with no nucleus or organelles, single circular ds DNA molecule (tightly condensed and called a nucleoid), no histones or other assoc. proteins. Replicate DNA in both directions from single point of origin ("theta replc.")
How do they reproduce?
By binary fission (chromosome replicates, cell divides into two cells: each cell bearing one chromosome); lacks nucleus => lack microtubules, spindle, centrioles.)
Plasmid that can incoporate into bacterial chromosome
Short, circular DNA outside chromosome (carry genes that are beneficial but not essential for survival and replicate independently)
donor produces a bridge (pilus) and connect to recipient; send chromosome/plasmid to recipient and recombinant can occur; F plasmid allowing pilus to occur; once recipient receives, it is now F+ and can donate as well. R plasmids provide bacteria with antibiotic resistance. Pili are also used for cell adhesion!
DNA is introduced into genome by a virus. When virus is assembled during lytic cycle, some bacterial DNA is incorporated in place of viral DNA. When virus infects another host, the bacterial DNA part that it delivers can recombine with the resident DNA.
What are R plasmids?
Provide bacteria with resistance against antibiotics
What are F plasmids?
Contains the genes that enable a bacterium to produce pili, so when a recipient receives the F plasmid, it too can become a donor cell
bacteria absorb DNA from surrounds and incorporate into genome.
Operon, function and consists of 4 things?
Function - control gene transcription and consists of
3. Structural genes
4. Regulatory genes
Exists only in prokaryotes
Do operons exist in prokaryotes?
Yes, they exist in prokaryotes, but they don't exist in eukaryotes
sequence of DNA where RNA polymerase attaches to being transcription.
region that can block action of RNA polymerase if occupied by repressor protein.
DNA sequences that code for related enzymes.
located outside of operon region, produces repressor proteins. Others produce activator proteins that assist the attachment of RNA polymerase to promoter region
E.coli is commonly found where?
Digestive tract of humans
Found in E.Coli and controls the breakdown of lactose
When lactose is available, the repressor will be bound to some lactose, rendering it inactive. This allows the RNA polymerase to transcribe the genes that code for the enzymes that break down lactose
since a substance (such as lactose) is required to induce (turn on) the operon, the enzymes that the operon produces are said to be inducible
(E. coli): produces enzyme for tryptophan synthesis; regulatory genes produce an inactive repressor => RNA pol produces enzymes. When tryptophan is available, no longer need to synthesize it internally: it binds to inactive repressor and activates repressor => able binds operator and block RNA pol. Tryptophan is corepressor.
as above, when structural genes stop producing enzymes only in presence of an active repressor. Unlike repressible enzymes, some genes are constitutive (constantly expressed) either naturally or due to mutation
Genes that are constantly expressed
What are the ways that eukaryotic gene expression is regulated?
1. Regulatory proteins
2. Nucleosome packing
3. RNA interference
Repressors and activators (like those in prokaryotes), influence how readily RNA polymerase will attach to a promoter region
In many cases, numerous activators are acting in concert to influence transcription
influences whether a section of DNA will be transcribed; if DNA segments are tightly packed by methylation, transcription will be more difficult
methylation of histones (tighter packing = preventing transcription); acetylation of histones (uncoiling and transcriptions proceeds). (side note: methylation also used in X-inactivation and on DNA bases to repress gene activity. Also, histone methylation usually prevents transcription, but sometimes it can activate it)
Methylation does what?
Of the histones, it will have tighter packing and prevent transcription
Of the histones, will uncoil and transcription will move forward
What is responsible for x-inactivation
Methylation and on DNA bases to repress gene activity
Note: it usually prevents transcription, but sometimes it can activate it too
When siRNA block mRNA transcription or translation or degrade existing mRNA
short interfering RNAS
block mRNA transcriptions (fold back within itself = dsRNA), translation, or degrade existing mRNA. siRNAs: dsRNA gets chopped up, then made single stranded. The relevant strand will bind to DNA (prevent transcription) or mRNA (signals destruction)
What makes up 97% of the human genome?
97% of human DNA does not code for protein product; noncoding DNA: regulatory sequences, introns, repetitive sequences never transcribed, etc. Tandem repeats abnormally long stretches of back to back repetitive sequences within an affected gene (e.g. Huntington's).
contains DNA segments or genes from different sources. DNA transferred from one part of the DNA molecule to another, from one chromosome to another, or from one organism to another
What causes recombinant DNA?
The transfer of these DNA segments can come from viral transduction, bacterial conjugation, transposons, or through artificial recombinant DNA technology. Crossing over during prophase of meiosis produces recombinant chromosomes.
What does recombinant DNA use to cut up DNA?
Where are restriction enzymes obtained?
From bacteria that manufacture the enzymes to combat invading viruses
How does the restriction enzyme usually cut?
The cut is usually staggered, producing fragments that have one strand of the DNA extending beyond the complementary -- sticky end
Sticky end results from
Restriction DNA cutting the DNA and creating a staggered cut
What is the purpose of restriction enzymes in bacteria? What are some examples?
used by bacteria to protect against viral DNA (protect their own DNA via methylation)
uses restriction endonucleases to cut up specific segments of DNA and left it with sticky end (unpaired).
To insert a foreign DNA fragment into another cell, the fragment is first introduced into another DNA molecule called
What is commonly used as a vector?
such as plasmid because DNA molecule used as a vehicle to transfer foreign genetic material into another cell.
How can a foreign DNA be introduced to a plasmid
To introduce foreign DNA into plasmid, the plasmid is treated with the same restriction enzyme so the same sticky ends to bind. DNA ligase stabilizes the attachments; then the plasmid is introduced into bacterium by transformation. Bacterium must be "made competent" to take up the plasmid (electroporation or heat shock+CaCl2)
How is bacteria "made competent" to take up the plasmid?
Electroporation or heat shock / CaCl2
What stabilizes the base pairing of the sticky ends?
By what method was human insulin introduced into E.Coli?
By using recombinant DNA technology and then introducing the recombinant plasmid into bacterium by transformation
Restriction fragments can be separated how?
By gel electrophoresis
agarose gel under an electric field for the separation of proteins based on charge and size (e.g. negative DNA moves toward positive anode from negative cathode). Shorter DNA moves further than larger; distributes DNA by size.
What can you do after electrophoresis?
DNA can then be sequenced, or probed to identify location of specific sequence of DNA
Can do on proteins too by adding SDS (denatures+linearizes+adds negative charge)
Electrophoresis is commonly used to do what?
Compare DNA fragments of closely related species in an effort to determine evolutionary relationships. Can also be done on proteins
For gel electrophoresis, DNA fragments are separated by what?
Molecular weight. The smaller ones will go faster
What's commonly used at crime scenes?
DNA fingerprinting, which uses RFLPs produced from DNA at a crime scene
Restriction fragment length polymorphism (RFLPs)
restriction fragments between individuals are compared, fragments differ in length are observed because of polymorphism (different length in DNA sequences).
Inherited in Mendelian fashion so often used in paternity suits, RFLP analysis used at crime scenes to match suspects.
What is RFLPs differing lengths due to?
DNA finger printing
RFLPs at crime scene compared to RFLPs of suspects.
Short tandem repeat (STR)
repeat of 2-5 nucleotides and different between all individuals except identical twins.
This is the type of DNA that lacks introns that suppress transcription
When foreign genes are inserted into the genome of a bacterium with recombinant DNA tech, introns only prevent their transcription so to avoid this problem, DNA fragment bearing the required gene is obtained directly from the mRNA that codes for desired polypeptide
Reverse transcriptase is used to make a DNA molecule directly from mRNA
introns often prevent transcriptions; this enzyme makes DNA molecule directly from mRNA. DNA obtained from this manner is complementary DNA (cDNA) which lacks introns that suppress transcriptions.
uses synthetic primer (the primer may be RNA or DNA oligonucleotides) to clone DNA (rapidly amplify). Taq polymerase (heat stable) + nucleotides + primers + salts (buffer) necessary.
Basically, billions of identical copies of genes are made within a few hours
Name of PCR machine
Thermocycler - as it warms and cools, hydrogen bonds break and separates the double stranded DNA. Taq polymerase binds to the primers on both strands and adds nucleotides.
What are the three steps of PCR?
1. Denaturation (>90C)
2. Primers + anneal (55c)
3. Elongation (Taq polymerase (70C)
Southern blotting vs Northern vs Western
Technique to ID target fragments of known DNA sequence in a large population of DNA. Electrophoresis fragments first, separate DNA strands (usually with NaOH) then transfer the SS DNA fragments to nitrocellulose membrane, then add probe which will hybridize and mark it.
S - DNA
N - RNA
O - O
W - Protein
Western - similar method for proteins: electrophoresis, blot to membrane, primary antibody specific to protein added to bind to that protein, then secondary anti-body-enzyme conjugate will bind to primary and mark it w/ enzyme for visualization..
Plasmids are circular dsDNA that have restriction sites (for restriction enzymes). Cut there à linear piece of DNA. Also has a promoter region + some gene product(s) (e.g. antibiotic resistance). We want to cut, add genes, close plasmid, add to something like bacteria to replicate it. Bacteria dislike plasmids à only fraction of them get taken up by some bacteria. Use antibiotic resistance gene to determine which bacteria were "transformed" that will survive on certain medium. Plasmid also has origin of replication. Plasmids will get reproduced during cell division or even not during cell division. If making a prokaryotic gene product in mammalian cell à need to add polyA tail for the mRNA to survive. If making eukaryotic gene product in prokaryotic cell à need to make sure no introns (use reverse transcriptase on the mRNA product to get the desired DNA fragment).
complementary BP's annealing
- Want to test for specific gene sequence in someone's DNA? Method 1 (single test only): take drop of blood, cut up DNA, use PCR method w/ specific primer for that region. If that gene is there à lots of copies. Gene not there à no copies. Method 2 (test for many things at same time): take drop of blood, PCR it to amplify. We have a solid support w/ pieces of ssDNA w/ specific sequence covalently attached à will hybridize to anything complementary (e.g. disease genes). On that same solid support we can put sSDNA pieces specific for other genes, can do this hundreds of times at different spots on this DNA microarray. Take desired amplified DNA, heat it to denature, add to DNA microarray, any DNA that hybridizes is a match. The amplified DNA we added is already fluorescently tagged à hybridization wash to get rid of weakly bound sequences (e.g. not a complete match) àadd a dye that will show heavily if something has bound to our microarray. Can also do this starting w/ mRNA à reverse transcriptase à PCR amplify à etc.
What marks proteins for degradation via proteasome
Ubiquitin and not ubiquinone
Not mature cells that dedifferentiate and can give rise to any and all human cells, and even an entire functional organism
Give rise to all tissue types, but not entire organism
Give rise to a limited range of cells within a tissue type
The branch of technology that produces new organisms or products by transferring genes between cells
A _____ enzyme recognizes a short DNA sequence, restriction site, and cuts both DNA strands at _____ sequences. The resulting double stranded restriction fragments have at least one single stranded end called ______ ______. These short extensions allow _____-bonded base pairs between the sticky ends of a _____, a cloning vector, and the DNA molecule of interest cut with the same ____ enzyme. When DNA ligase permanently seals the two fragments, a recombinant DNA is produced
What is this process? When the host cell is grown into a culutre to form a clone of cell containing the cloned gene of interest, such as when technology is used to produce insulin when inserted into E.Coli
Insulin (lowers blood sugar levels) can be made for medical purposes by bacterial. Meaning that bacteria can be induced to use the universal DNA code to transcribe and translate a human gene.
Heat shock method
During transformation, the plasmid contains a gene for antibiotic resistance, so transformed bacteria will be able to grow on a medium that contains antibiotics whereas bacteria without the plasmid will die. This allows us to safely mass produce important proteins used in medicine and also has an important role in gene expression
Human Genome Project
1990 - publicly funded / int'l - determined to sequence every chromosome, base by base, genome by genome. Can be used to study evolutionary history, genetic diseases, and make pharmaceuticals
During posttranslational modification, the polypeptide from the eukarytopic cell typically undergoes substantial alteration that results in
the polypeptide undergoing a conformational change
Unprocessed RNA molecules that are precursors
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Core Topic 2 Molecular Biology | IB Biology Guide
DAT BIO: Molecular Genetics
biology chapter 12 and 13 DNA and RNA
OTHER SETS BY THIS CREATOR
d4: signaling II (cyclic-amp, g-proteins, and ADP-ribosylations
d4: gram + cocci
d4: antimicrobial drugs II (protein synthesis inhibitors)
d3: signaling I
THIS SET IS OFTEN IN FOLDERS WITH...