MIDTERM 1 Flashcards
Liter
L= 1 known conversion factors
Milliliter
mL: .001L, 1 x 10^-3 L.
One thousandth of a liter
Microliters:
P2
P20
P200
P1000
P2: .2-2 ul
P20: 2-20 ul
P200: 20-200 ul
P1000: 200-1000ul
Stock solution
A concentrated for of a reagent that is often diluted. nX n>1. Given names that correspond to how concentrated they are.
Conversion Units
M,m,X,%, and some unit over another unit (g/l, mg/ml,etc.)
DNA
A complex molecule containing the genetic information that make up the chromosomes
carrier of genetic information
mRNA
messenger RNA; type of RNA that carries instructions from DNA in the nucleus to the ribosome
mRNA happens in nucleus
pre-mRNA
-occurs in transcription
-Immature mRNA; the first strand of mRNA produced in transcription
that contains both introns and exons
tRNA
-Transfer RNA provides linkage between mRNA and amino acids and transfers amino acids to ribosomes
- present in the cytoplasm
-Transfer amino acids to match the correct mRNA codon
INtrons
“stayes IN the nucleus”
-non-coding region of DNA
-does not code for protein
EXons
“EXIT/Expresses”
- expressed sequence of DNA
- codes for a protien
rRNA
-Type of RNA that combines with proteins to form ribosomes
- occurs in cytoplasm
Spliceosome
Complex of enzymes that serves to splice out the introns of a pre-mRNA transcript this releases the introns and join the two exons
Splicing
proccess of removing introns and reconnecting exons in a pre-mRNA
- removal of introns
- occurs in nucleus
Ribosome
- Cytoplasmic organelles at which proteins are synthesized.
-the “body” that makes proteins
- Uses the amino acids made by tRNA to create proteins that will have a certain task
cDNA
-“copy DNA”
-ONLY EXONS
-“complementary DNA”
-Synthesized in a lab
-it is produced by synthetically doing reverse transcription of mRNA. Because of eukaryotic mRNA splicing, cDNA contains no introns
gDNA
-“genomic DNA”
-the original, hard copy of genetic material for a cell
- contains both exons and introns
- found in body
Reverse Transcription
- Synthesis of DNA from an RNA template
(RNA—–>DNA)
Reverse Transcriptase
-Enzyme responsible for the formation of cDNA
-copy of RNA made through reverse transcriptase
-the DNA copy synthezised by reverse transcriptase is cDNA
- cDNA serves as a template for PCR
Protein
- an organic compound that is made of one or more chains of amino acids (codon) and that is a main component of all cells
- A set of specific nucleotides encodes for a specific protein
DNA Polymerase
-Enzyme invovled in “DNA replication” that joins individual nucleotides to produce a DNA molecule
-enzyme that synthesizes new copies of DNA
-Needs a primer
RNA Polymerase
Enzyme that links together the growing chain of RNA nucelotides during ‘transcription’ using a DNA strand as a template
-enzyme that makes RNA from DNA template
-Doesn’t need a primer
Polymerase chain reaction (PCR)
A technique that amplifies DNA of specific genes
- PCR includes to incubate with specific primers, DNA polymerase molecules, and nucleotides
PCR step 1
Denaturing 98°C
- Heat up to break apart double-stranded DNA molecules
PCR step 2
Annealing 50-70°C
- cooled down to allow primers to bind to each DNA strand on opposite ends of the segment in order to be copied
PCR step 3
Extension 72°C
-TAQ polymearse synthesizes the first set of cDNA to form new strands
- makes polymerase extend to 3’ to 5’
Polymorphism
If two individuals have sequence differences at the same place in the genome
Insertion Deletion (indel)
When 1 or more nucleotides of DNA is present in one individual and absent in another
*it is represented as dashes in programs
Single Nucleotide Polymorphism (SNPs)
Difference at one nucleotide in a DNA sequence among the genome sequence
- sickle cell anemia were a SNP caused a change of glutamic acid to valine
EX: A is changed to G
Transposable Element
A segment of DNA that can move spontaneously within or between chromosomes. A mobile genetic sequence
- can cause mutations
BLAST
Helps analyze DNA sequence
Blast Hit
Bioinformatics page that allows us to see the sequence of nucleotides within a DNA sample. We can see introns and exons and specific base pair length
Multiple sequence Alignment
the alignment of three or more biological sequences (proteins or nucleic acid) of similar length
we used the program MUSCLE
Primers
Short segments of DNA that guide DNA polymerase to the section of DNA to copy
-main job is to bind to bases
-In our experiment, the priers bind around the Actin gene
- 1 Forward and 1 Reverse
Taq
-Short for taq polymerase; a heat stable DNA polymerase that is used during PCR to synthesize new DNA strands
- it can withstand high temperatures of PCR
Negative control
Control group where conditions produce a negative outcome. Negative control groups help identify outside influences which may be present that were not accounted for when the procedure was created
-ddH2O
-typically water
Master Mix
Special reaction buffer with all the components needed for PCR to occur.
- free nucleotides, TAQ polymerase, primers, and reaction buffer
-Needed for new strand of DNA to be synthesized
-added before temperature change
Gene expression
The process by which information encoded in DNA directs the synthesis of proteins or, in some cases, RNAs that are not translated into proteins and instead function as RNAs
Antibiotic Selection
Used to recover transformants. Scientists use antibiotic resistance genes as genetic markers to identify which bacteria were transformed
Plasmid
A small ring of DNA that carries accessory genes separate from those of the bacterial chromosomes
the genes carried in plasmind provide bacteria with genetic advantages such as antibiotic resistance
Bacterial Transformation
Ability of bacteria to alter their genetic makeup by “up taking foreign DNA” from another bacterial cell and incorporating it into their own
Constitutive Promoter
“Active all the time”
Housekeeping cell
The RNA polymerase binds to the promoter, which then allows transcription to occur
Conditional Promoter
“Requires a Condition to turn on”
In order for RNA polymerase to bind to the promoter and initiate transcription, a transcription factor must attach to it; hence, a sugar or hormone must be present. The transcription factor will bind to the sugar or hormone, enabling the RNA polymerase to bind to the promoter and initiate transcription.
Synonymous
-Silent mutation
-does not change the amino acid, therefore does not change the protein
-Protein function is retained
Non-Synonymous
-A mutation in a gene that changes the amino acid sequence of the protein that gene encodes
-Detrimental to a protein function
Missense
A DNA change that results in a different amino acid
Nonsense
A DNA change that results in a premature “STOP” codon
-the most detrimental SNP effect
Identifying the number of reactions
(# of given samples) + 1 Negative control (water) + 1 positive control (master mix)
Competent
Plasmid mixed with bacterial cells that have been treated with calcium chloride to make them capable of taking up DNA
Tranformants
Cells that take up plasmid
Why is GFP userful?
Visualizing the presence of the gene doesn’t require sacrificing the tissue to be studied.
What is the correct description of the steps of PCR in correct order from first to last:
- Double helix is melted to singe strands
- Primers bind to template DNA
- Primers are extended by DNA polymerase to make new strand
What is used as the negative control in the PCR experiment?
Water
How much agarose is requires to make a 2% gel with a volume of 200mL 1X TAE?
4g
The key term ‘Polymorphism’ most closely means what when broken down?
Many forms
What does PCR stand for?
Polymerase Chain Reaction
T/F: the purpose of the DNA polymorphism experiment is to look for genetic differences among individuals of different species
False
*looking for differences among SAME species
A PCR reaction experiment that includes 1 unknown DNA sample and 2 controls (positive and negative) you will need how many total reactions in the Master Mix?
4
*always add one more in case of pipet errors
Match the following reagents with the correct component of the lab they were use in (the master mix or gel of electrophoresis):
- DNA Polymearse
- DNTPs (nucleotides)
- Ethidium Bromide (EtBr)
- Agrose
- Tris-Acetate EDTA buffer (TAE)
- Primers
- DNA Polymearse: Master Mix
- DNTPs (nucleotides) : Master Mix
- Ethidium Bromide (EtBr): GEL
- Agrose: Gel
- Tris-Acetate EDTA buffer (TAE): GEL
- Primers: Master Mix
What does the “Denature” step of PCT refer to?
Double helix DNA strands is heated and separated into single strands
gDNA is a copy of____, while cDNA is a copy of ____
Genomic DNA, mRNA
Of the choices below, what is the worst combination of SNP type and gene location?
- Nonsynonymous SNP in an exon.
- Synonymous SNP in an intron.
- Synonymous SNP in an exon.
- Nonsynonymous SNP in an intron.
- Nonsynonymous SNP in an exon
What organelle transfers amino acids to the growing protein in the cytoplasm via translation?
Ribosome
DNA molecules have a ____________ charge, so larger DNA molecules will travel _____________ through the agarose gel compared to smaller DNA molecules. [HINT: The gels run from the black wire (negative anode) to the red wire (positive anode)
negative, a shorter distance
How does translation stop adding amino acids to the growing protein?
There is a stop codon in the mRNA
You run PCR and gel electrophoresis examining a gene where the expected band length is 1500 nucleotide base pairs (bp). Which of the following scenarios would show two bands in one lane on the agarose gel. Select ALL that apply. (Allele = one version of a gene)
- Two different alleles, one with a synonymous SNP compared to the other allele.
- Two different alleles, one with a 500 bp insertion in an intron in the gDNA compared to the other allele.
- Two different alleles, one with a nonsynonymous (missense) SNP compared to the other allele.
- Two different alleles, one with a 500 bp insertion in an exon compared to the other.
- Two different alleles, one with a nonsynonymous (nonsense) SNP in an exon early in the gene compared to the other allele.
- Two different alleles, one with a 500 bp insertion in an exon compared to the other
This transcribes the DNA into pre-mRNA
RNA polymerase
Has a complementary sequence of mRNA and carries an amino acid to the new growing protein
tRNA
Non-coding region removed during splicing
Intron
Contains introns and exons before splicing
pre-mRNA
What is the correct order of steps in PCR?
Denaturing–>Annealing–>Extension
What animals was GFP originally clored from
Jelly fish
Which of the following molecules and processes of genetic information transfer occur in Nucleus?
1.Translation
2.Transcription
3.Splicing.
4.tRNA
- Transcription
- Splicing
T/F: During thr 3 steps of PCR, the temperature gets colder before it warms up again.
True
What is incorrect about gel electrophoresis?
- DNA fragments that are very small appear at the top of the gel
- Gel electrophoresis along can be used to detect SNPs
To be able to clone GFP for expression in a new organism than the original, introns need to first be removed, what type of molecule did the scientists clone?
cDNA
Competent cells are fragile, which means that you should not do what:
- Centrifuge
- Pipette them up and down
3.Vortex
Describe how conditional gene expression functions.
Controls when the gene of interest is expressed as protein
Why are competent cells used in bacterial transformation?
Competent cells are capable of taking up plasmids
Which of the following statements about setting up a PCR reaction is incorrect? Select all that apply.
- The number of reactions of master mix prepared should equal the number of samples
- The C1V1=C2V2 equation should be used to determine what volume of MyTaq is needed for the reaction.
- Template for all samples should be added to the master mix before aliquoting into strip tubes
- After all components are added to create the mastermix it should not be mix
1,3,4
What is the name of the molecule that turns off a gene by preventing mRNA polymerase from binding to the promoter?
Transcription Factor
What volume of a 75% stock solution is needed to make 10 µl of a 12% working solution, and how much water will need to be added to the stock?
1.6 µl of stock, and 8.4 µl of water
What volume of stock and water is required to make 500 µl of a 2X solution starting with 10X stock?
100 µl of stock and 400 µl of water
Solve:
ddH2O ?
Forward Primer 0.5
Reverse Primer 0.5
2xMytaq ?
Subtotal ?
DNA 5.0
Grand total 20.0
Solve:
ddH2O 4.0 24.0
Forward Primer 0.5 3.0
Reverse Primer 0.5 3.0
2xMytaq 10 60
Subtotal 15 90
DNA 5.0 ——
Grand total 20.0 ——–
Nymphia needs to make 50 mL of diluted TAE solution for
her experiment. The prep room has 100 mL of 1X (M) TAE
stock solution. What concentration of TAE will Nymphia
need to prepare?
2X
