bio_142 unito uno

when & what did Frederick Griffith find out ???

non-pathogenic bacteria can be transformed to Pathogenic

1928

when & what did Avery, McCarty, & MacLeod find out ???

the transforming substance is DNA

1944

when & what did Erwin Chargaff find out ???

Reported that DNA composition varies from one species to another, but A=T and G=C.

1950

when & what did Alfred Hershey and Martha Chase find out ???

DNA is the genetic material on phage

1952

what happens in the 1928, Transforming Principle ???

imagen

what happens in the 1952, DNA is genetic material in T2??

imagen

what are the components of a nucleotide?

1. nitrogenous base

2. phosphate group

3. sugar

   1. (deoxyribose for DNA & ribose for RNA)

differences between the pentose sugars ??

• deoxyribose for DNA (just h, NO oxygen)

• ribose for RNA (OH plus oxygen !!)

the 4 (or like 5) nitrogenous bases ??

• thymine (T)

• cytosine (C)

• adenine (A)

• guanine (G)

• uracil (U) (RNA)

what are the aspects or whatever of the pyrimidines nitrogenous bases?

pyrmidines

• thymine & cytosine (prob also uracil)

• one hexagon

what are the aspects or whatever of the purinesnitrogenous bases?

purines

• adenine & guanine

• two joint hexagon

primary structure of DNA & RNA??

• Polynucleotide Strands

the bond uniting DNA & RNA ?

phosphodiester bond

what is DNA secondary structure ?

Double helix interacting by two forces:

1. Hydrogen bonds

2. Interactions between stacked base pairs

what is RNA secondary structure ?

• complex secondary structure

• hairpin

• stem

What does it mean to “manipulate” DNA?

1. cut DNA (think restriction enzymes)

2. separate & visualize DNA (gel electrophoresis)

3. Quantify DNA (qPCR)

4. Combine DNA / Recombinant (gene cloning)

how-to Cut DNA

• restriction enzymes

how-to Separate & Visualize DNA

• gel electrophoresis

how-to quantify DNA

• qPCR

  • plots the amplification curve compared with a standard

how-to combine DNA (recombinant)

• gene cloning

what does PCR stand for?

Polymerase Chain Reaction

what does PCR do?

• amplify DNA

steps of PCR

Changing and Cycling temperatures (in the correct order):

• Denaturing (90-100ºC)

• Annealing Primers (30-65ºC)

• Extending the molecule (72ºC)

how to determine DNA Sequencing

• Dideoxy Sequencing (Sanger Method)

• Next Generation Sequencing (Illumina)

Deoxyribonucleotide vs Dideoxyribonucleotide

Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3′-OH group, which terminates DNA synthesis

Structure of deoxyribonucleoside triphosphate, the normal substrate for DNA synthesis.

dATP, dTTP, dGTP, dCTP

Structure of dideoxyribonucleoside triphosphate, which lacks an OH group on the 3′-carbon atom.

ddATP, ddTTP, ddGTP, ddCTP

what does CRISPR-Cas9 ?

• Cut & Edit DNA

what is CRISPR and what are its step??

• DNA: “Clustered Regularly InterSpaced Palindromic Repeats”

• Protein: Cas-9

• Phase 1 - immunization / acquisition

• Phase 2 - defense / resistance

all about that…Cas9 enzyme

• protein

• a bacterial endonuclease protein that forms a double-strand break (scissors) at a specific site within a larger recognition sequence, or target site.

• the Cas9 recognition sequence includes a 20- nucleotide target sequence that is determined by a guide RNA (also called single-guide RNA), below in red, bound to the enzyme

Single Guide RNA (sgRNA)

A form of guide RNA that forms a complex with Cas9.

The sgRNA is approximately 100 nucleotide–long fusion

and has two regions :

• Guiding region —a typically 20-nucleotide region that

is complementary to the target DNA sequence and that

defines where Cas9 cuts

• Scaffold region — a region that forms a hairpin loop

structure that binds tightly in a crevice of the Cas9

protein. The sequence of this region is typically the same

for all sgRNAs.

Protospacer-Adjacent motif (PAM) Sequence

A sequence motif immediately downstream of the target sequence is required for Cas9 function. Cas9 recognizes the PAM sequence 5'-NGG-3' where N can be any nucleotide (A, T, C, or G).

what are the 4 steps of gene editing ?

1. Targeting

   1. Cas9 recognizes any random three-nucleotide sequence PAM

2. Binding

   1. Cas9 binds and unwinds the DNA helix upstream of PAM. If the DNA sequence matches the sgRNA sequence, then the RNA and complementary DNA will bind to form a DNA-RNA double helix.

1. Cleaving

   1. Cas9’s nuclease activity is activated. It cuts both strands of the DNA three nucleotides upstream from PAM.

1. DNA Repair

   1. Repair enzymes fix the break in the DNA. If a homologous template is NOT present, the cell repairs the break using nonhomologous end joining (NHEJ). If a homologous template IS available, the cell repairs the break using homology-directed repair (HDR)

what is the PAM nucleotide sequence ???

5’-NGG-3’

What is transmitted? in transmission genetics ?

Cellular, molecular (genotype)

How is it transmitted? in transmission genetics ?

Cellular (cell division)

What is the product of transmission? in transmission genetics ?

Molecular pathways, products (phenotype)

Monohybrid? meaning

• Heterozygotes with one gene of interest

Dihybrid? meaning

• Heterozygotes with two genes of interests

what are the loci doing usually in dihybrid alleles ?

• Loci are unlinked

General Assumptions of… simple inheritance

• One characteristic (e.g., flower color)– one gene (locus) – akamonogenic.

• One gene – has only two possible alleles controlling two contrasting traits (e.g., purple vs white)

• Complete dominance

what does that actual chi-square statistic mean?

probability that the difference between the observed and the expected values is due to chance alone.

what’s that degree of freedom (df) ?

the number of ways in which the expected classes are free to vary

null hypothesis

_____ produces EQUAL AMOUNTS of ______

alternative hypothesis

_____ does NOT produce EQUAL AMOUNTS of ______

formula for chi-squared ??

(O-E)² / E

(observed - expected)² / expected

How to interpret P-values?

• probability that the difference between the observed and the expected values is due to chance

• If p value > 0.05; greater than 5% probability that the deviation between observed and expected is due to random chance. Null hypothesis is not rejected.

how to really interpret the final chi-squared decision ?

There is ____ evidence to reject the null hypothesis, and thus the differences have a —-probability to be due to chance

general assumptions about… simple inheritance !

• one characteristic = one gene (locus)

  • monogenic !!

• one gene = only 2 possible alleles

• complete dominance

• its dihybrid ! (loci are unlinked)

what does dihybrid mean ?

loci are unlinked

what did HERMAN HENKING do and when?

• x-bodies with the firebugs!

• 1891

what did CLARENCE E MCCLUNG do and when?

• finds that x-body is a chromosome (grasshoppers!)

• 1899

what did NETTIE STEVENS & EDMUND WILSON do and when?

• discovered Y-chromosomes and their relation to sex determination (mealworms!)

• 1905

what did THOMAS H. MORGAN do and when?

• found that genes are on chromosomes and sex-linked genes exhibit unique patterns of inheritance!

• FRUIT FLIES

  • traits only expressed on certain genders; only males have white eyes

• 1910

what did CALVIN BRIDGES do and when?

• nondisjunction hypothesis !

• with fruit flies, sometimes things don’t split equally and there are unusual phenotypes

  • ex) white eyed females with X^wX^wY

• 1913

what are those systems of chromosomal sex determination /w examples ?

• XY system: ♀ - XX ♂ - XY

  • ex) humans and mammals

• ZW system: ♀ - WZ ♂ - ZZ

  • ex) birds!

• XO system: ♀ - XX ♂ - XO

  • ex) grasshoppers! (the o stands for zero)

• haplo-diploid system: ♀ - 32 chromosomes (diploid) ♂ - 16 chromosomes (haploid)

  • ex) bees!

dioecious means?

• male and female reproductive structures in the different organisms

• ex) chromosomes, temperature

monoecious means?

• both male and female reproductive structures in the same organism

examples of sex-linked (X-linked) traits

• color blindness

• duchenne muscular dystrophy

• hemophilia

details on the Lyon hypothesis ?

• by Mary Lyon

• X-inactivation happens! and its random

• the unactivated chromosomes become “barr bodies”

what are barr bodies ?

• the non-functional inactivated X chromosome

these are the sex-influenced characteristics… ?

• determined by autosomal genes

• inherited according to Mendel’s principles

• expressed differently in males and females (dominant vs recessive)

• ex) Bb is dominant in males & Bb is recessive in females

• ex) male-patterned baldness

these are the sex-limited characteristics… ?

• determined by autosomal genes

• expressed in only one sex (both can have the genes tho)

the steps to ovary development ???

• NR0B1 gene →

• transcribed to RNA →

• RNA is translated into DAX protein →

• OVARY DEVELOPMENT

the steps to testis development ???

• SRY gene →

• transcribed to RNA →

• RNA is translated into TDF (testis-determining factor) protein →

• testosterone produced →

• ANTI-MULLERIAN HORMONES →

• no DAX so no ovaries

what is that X-chromosome important reproductive gene ?

• NROB1 (nuclear receptor 0B1)

what is that Y-chromosome important reproductive gene ?

• SRY (sex-determining region of Y)

what does the NROB1 gene do ??

• Encodes DAX protein

• Ovary development

what does the SRY gene do ??

• Encodes TDF (testes-determining factor) / TRY proteins

• Testosterone and Anti-Mullerian hormones production

• Cancels action of DAX

what exactly is… nondisjunction ?

• unequal division of chromosomes during cell division !

• unnormal gametes at the end of the process

what’s the details on the amount of chromosomes in the human body ?

• 46 total

• 23 pairs

  • 22 autosomal; 1 sex

who the hell is mary lyon ?

• X-inactivation lady

• 1961 !

• also described barr bodies

what happens during crossing over for the sex chromosomes ?

• they don’t really cross over fr

• its not super favored but it does still happen only like very occasionally

why is X-inactivation important ?

• no need for a “double dosage” of genes

• they’re like the same genes but twice

what is the charge of genetic material ??

• lowkey trick question

• DNA is negatively charged but technically they didn’t know this about “genetic material” back in the day

what’s the charge of DNA?

• NEGATIVE CHARGE

what the hell is oogenesis ?

• meiosis but in the eggs during like fertilization, i guess

• you get one ovum and no polar bodies

which sugar ??? ಠಿ_ಠ

• ribose !

which sugar ??? ಠಿ_ಠ

deoxyribose!

which base ??? ಠಿ_ಠ (plus type)

THYMINE (T)

• pyrimidine!

• has that methyl CH₃ group!!

• meTHyl group

which base ??? ಠಿ_ಠ (plus type)

CYTOSINE (C)

• pyrimidine!

• has that amino NH₂ group!!

• only 1 O bond, cytosine only has 1 O

which base ??? ಠಿ_ಠ (plus type)

URACIL (U)

• pyrimidine!

• has NO outward functional group!!

• U need that O₂ (2 double bonded oxygen

which base ??? ಠಿ_ಠ (plus type)

ADENINE (A)

• purine!

• has that amino NH₂ group!!

• Add that Amino

which base ??? ಠಿ_ಠ (plus type)

GUANINE (G)

• purine!

• has that amino NH₂ group plus double bonded oxygen!

• GO get that Oxygen

what is the temp for and the FIRST step of PCR ?

• Denaturing (90-100ºC)

what is the temp for and the SECOND step of PCR ?

• Annealing Primers

  (30-65ºC)

what is the temp for and the THIRD step of PCR ?

• Extending the molecule (72ºC)

look at this PCR graph

..

dideoxy sequencing also known as….

• Sanger Method!

nest generation sequencing also known as….

• illumina !

limitations of CRISPR ?

• off-target cleavage of DNA!

• potential mutations if DNA repair does not go well

in CRISPR, what does the cas-9 enzyme serve as ?

• a sort of molecular scissor

in CRISPR, what does the guide RNA serve as ?

• programmable GPS that tells the cas-9 (molecular scissors) where to cut

in CRISPR, how long is a guide RNA?

like 20 nucleotides

in CRISPR, what does the target sequence need to be near to for everything to occur?

• a PAM motif!

what’s the 5’ to 3’ sequence of the PAM motif?

• 5’-N-G-G-3’

in CRIPSR, what happens if the target sequence is not a match of the guide RNA?

• cas-9 disengages and the DNA zips back into a double helix

in CRIPSR, what happens if the target sequence is a match of the guide RNA?

• the guide RNA base pairs with the complementary DNA sequence, forming a DNA-RNA helix

in CRISPR, what does cas-9 do when activated ?

• makes specific cuts in DNA at a position 3 nucleotides upstream from the PAM site.

in CRISPR, how far upstream of the PAM does cleavage happen?

• 3 nucleotides upstream