Dynamic Genome Midterm 1

what are the SI Prefixes

Pipette Review

Dilution Equation

water= final working volume - initial stock volume

PCR Table calcuations

7.5µl of 2X MyTaq

1.5µl water

for 4 RXNS:

2x MyTaq → 30µl

H20 → 60µl

What is the purpose of PCR 

amplify a certain part of DNA to be able to have a quantity of DNA that can be studied 

what are the stages if PCR?

denaturing: heat breaks hydrogen bonds within the double stranded DNA base pairs, seperating into 2 single DNA strands

annealing: temp is lowered to let DNA primers bind to their complementary sequence, thus they are attached ready for extension

extension: temp is raised again to a optimal working temp for the Taq polymerase, the DNA polymerase binds to a primer and starts synthesising a complementary DNA sequnce in the 5’→3’ direction

this will happen for as many cycles as needed where the DNA is doubled each cycle

What materials are needed for PCR

2x MyTaq

Forward & reverse primers →short, single stranded DNA sequences that primes ssDNA so DNA polymerase knows where to bind and start elongating for annealing

ddH2O

Positive and negative control. Positive control is used to verify the PCR is working using a well known sample and negative control verify that the mastermix is not contaminated w/DNA

What does MyTaq Contain? 

Taq polymerase (DNA polymerase of extension)

Nucleotides for the polymerase to use during extension 

Buffer to provide good environment for DNA polymerase to work effectively 

Gel Electropheresis

DNA polymorphism

variation of specific DNA sequences 

SNPs

A single nucleotide polymorphism: a difference in 1 nucleotide

sickle cell anemia is bc of a SNP

error during DNA replication

2 types of SNPs:

Synonymous: silent (doesnt change the amino acid that was coded for in the codon)

Nonsynonymous SNP: Missense/nonsense(most severe mutation) mutations that does change amino acid sequence

Indels

insertion/deletion of 1 or more nucleotides of DNA 

can cause frameshift mutations if the number of necletides inde;ed is not a multiple of 3 

“-” gaps 

TEs

Compare the 3 polymorphisms

Gene Expression 

process of transcribing and translating DNA→protein 

transcription:

• in the nucleus, DNA → pre-mRNA →mRNA 

  • RNA polymerase, spliceosomes

Translation

• in cytoplasm, mRNA → tRNA → amino acid chain

  • ribosome

transcription and translation steps 

Reverse Transcription

allows synthesis of DNA (in cDNA form) from mRNA using reverse transcriptase

• cDNA is intron-less version of gDNA since it is based on the spliced mRNA

  • allows us to determine what was spliced out of mRNA and the difference in sequence length

  • more stable than mRNA

  • cDNA are often shorter →further downstream in gel electrophoresis

Sequence analysis (MUSCLE & BLAST)

experiment 3

Regulation of gene expression

Constitutive: genes that are always on

conditional: genes that are conditionally on

Transformation

inserting DNA into living organism, the organism can take up the DNA DNA as its own and express into protein

Plasmid

A circular strand of DNA that can be taken up by the bacteria, bacteria can express the genes located in the plasmid as their own DNA

• genes added to the plasmid must be cDNA/intron-less because bacteria cant slice out introns

reporter gene

a gene that indicates gene expression is occuring (ex GFP)

Selective gene

gene that ensures only transformed bacteria are left alive (AmpR)

constitutive expression process

Conditional Expression process

RNA polymerase cannot bind to promoter because a transcription factor is already binded

sugar/hormones if it binds to TF then it comes off from the gene and the RNA polymeras can bind to the promoter

how to calculate DNA copies per PCR cycle

2 ⁿ (x) 

n= # of cycles

x= # of starting cycles 

Translation

in cytoplasam mRNA meats with ribosomes where it contains both proteins and rRNA

Which of the following is needed for DNA amplification to occur?

A) Ribonucleotides

B) RNA primers

C) DNA polymerase

D) Both B and C

E) All of the above

cDNA polymerase

What does PCR stand

Polymerase Chain Reaction

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 725 µl of a solution with a concentration of 55 µg/µl when starting with a stock concentration of 275 µg/µl?

0.145 mL of stock and 0.580 mL of water

A. Two different alleles, one with a synonymous SNP compared to the other allele.

B. It has no significance.

Incorrect answer:

C. Two different alleles, one with an SNP compared to the other allele.

D. A heterozygous allele which appears as two distinct bands

D. A heterozygous allele which appears as two distinct bands

In a PCR reaction which of the following is required for DNA amplification to occur?

A. RNA primers

B. RNA primers and (Taq) DNA polymerase

C. Ribonucleotides

D. (Taq) DNA polymerase 

D. (Taq) DNA polymerase 

gDNA is a copy of ________, while cDNA is a copy of _________.

A. genomic DNA, mRNA

B. genomic DNA, tRNA

C. rRNA, genomic DNA

D. mRNA, genomic DNA

D. mRNA, genomic DNA

In a PCR reaction which of the following is required for DNA amplification to occur?

(Taq) DNA polymerase 

tRNA

Has a complementary sequence of mRNA and carries an amino acids to the growing protein chain

RNA Polymerase

Has a complementary sequence of mRNA and carries an amino acids to the growing protein chain

The following question assumes the same transformation protocol that was used for the experiment you conducted in lab.

What would happen if you took some of the E. coli bacteria from the 'LB + ampicillin plate' and spread the bacteria on a new plate with 'LB + ampicillin + galactose'? (alive/dead & glow/no glow)

The bacteria will be dead.

The bacteria will be alive but won't glow under

The bacteria will be alive and will glow under UV light.

The bacteria will be alive but won't glow under UV light.

Which of the following templates and primer pairs could you use to detect a large TE insertion in Exon 3? Select ALL that apply.

gDNA: F1 and R1

gDNA: F1 and R2

cDNA: F1 and R2

cDNA: F2 and R2

cDNA: F1 and R1

If an 'Indel' polymorphism occurred on the original 'TAT' codon, which of the following choices could be observed? Select ALL that apply. (' - ' indicate gaps)

TAT —> TA<CAGTG ... CACTG>T (inverted repeat)

TAT —> TAG

TAT —> T - T

TAT —> TAAT

identify snps

where is indel

what is in mytaq

taq polymerase, nucleotides, buffer

t/f an indel can cause a frameshift

true

t/f indels are reversible 

true 

what is the order of gene expression

Transcription, splicing, translation

how do we get cDNA from gDNA

reverse transcription of mrna

What is the difference between MUSCLE and BLAST?

BLAST shows difference between gDNA and CDNA, MUSCLE shows polymorphisms

transformation efficiency formula

1.) calculate the mass of plasmid used

• #ng of plasmid = (volume of DNA used)(concentration of DNA)

2.) calc the transformation efficiency

• transformation efficiency (cfu/nzg)= (#colonies/#ng of plasmid) x ( total volume µl/ plated µl)

3.) change to cfu/µg 

• cfg/ng x 1000 ng/µg = cfu/µl