HLSC322 exam 3 woohoo yippee

Topics: Molecular methods: Genetic and -omic analyses, Chromosomal variation, Regulation of prokaryotic gene expression

Restriction enzyme

One of bacteria’s antiviral defense mechanisms

dsbreaks DNA at specific ‘palindromic’ sequences

eg. EcoR1’s recognition sequence:

5’ – GAATTC – 3’ 

3’ – CTTAAG – 5’

^ reading 5’ to 3’ on both strands gets u the same sequence

Sticky vs. Blunt cuts

Sticky = staggered = cohesive cuts will leave overhangs, while blunt cuts do not

3 features of a cloning vector

Origin of replication recognized by bacteria

• so recombinant plasmid can get replicated

Selectable marker (often antibiotic resistance)

• allows isolation of transgenic bacteria from nontransgenic bacteria

Polylinker aka Multiple Cloning Site (MCS)

• contains many restriction sites — allows piece of DNA to be inserted into that region

Steps of DNA insertion into vector

1. Digest plasmid

2. Phosphatase treatment

3. Digest DNA of interest

4. Purify both fragments

5. Allow fragments to anneal

6. Seal nicks in bonds

7. Transform bacteria

8. Spread colonies on plate with ampicillin

9. Isolate colonies

10. Determine if correct vector was inserted into correct vector

Screening for success of DNA insertion - Disruption of LacZ

So u got ur Plasmid with LacZ and restriction site within the LacZ 

If u place untreated bacteria in media with ampicillin and X-gal, they appear blue — this is cuz LacZ gene codes for beta-galactosidase, which cleaves x gal to produce blue pigment.

When ur DNA of interest gets inserted in middle of LacZ, it effectively kills the gene: so DNA successfully inserted → colony remains white.

Polymerase Chain Reaction

• what are the ingredients?

• what is the procedure?

Technique to amplify — produce many many copies of — DNA

Ingredients: heat-resistant Taq polymerase, dNTPs, primers, thermocycler machine

• since primers are used, u must have some knowledge about sequence u are PCRing

Procedure

• Heat to 90 - 100^oC to separate 2 strands

• Cool to 30 - 65^oC so primers can anneal

• Heat to 60 - 70^oC so Taq polymerase can do its thing

(exact temperatures depend on specific sequence, prolly more GC% content means higher melting point)

Types of genomic differences between hoomans

Single Nucleotide Polymorphisms (SNPs)

Indels

Short Tandem Repeats (STRs)

Copy Number Variants (CNVs)

Combined DNA Index System (CODIS)

uses standard set of 13 primers for 13 unlinked loci as a genetic profile

used for forensics, paternity tests and the like

Haplotype

group of closely linked genes / genetic markers on a single chromosome that tend to be inherited together

used to trace ancestry

Transposable elements

aka jumping genes / transposons / mobile DNA

44.4% of human genome

code for transposase enzyme. Transposase does staggered cut → transposable element inserts → DNA polymerase fills in gaps, forming flanking direct repeats

4 mechanisms of transposition

DNA transposition

Replicative transposition = new copy inserts itself, original copy stays behind

Nonreplicative transposition = original copy moves to new site

RNA intermediate transposition

How do genomes retain stability with so many transposable elements potentially messing things up?

• Methylation

• piRNAs

• Inhibition of transposase expression

Polymorphism

natural occurrence of multiple alleles of gene or DNA sequence within population

Allo vs. Auto polyploidy

having more than 2 complete sets of chromosomes from 2 or more different species (i.e. vs. the same species

more common in plants than animals

Aneuploidy & its 4 types (in humans)

Having different than 2 complete sets of chromosomes

Nullisomy = 0 copies (2 deleted)

Monosomy = 1 copy (1 deleted)

Trisomy = 3 copies (1 duplicated)

Tetrasomy = 4 copes (2 duplicated)

Primary vs. Familial Down Syndrome

Primary DS (95% of cases) = Trisomy 21 ft. meiotic nondisjunction

Familial DS = Parent carries a balanced rearrangement of chromosome 21 and another chromosome (e.g., 14), which does not affect them but leads to unbalanced, inheriting three 21st chromosome copies in their child.

4 types of major chromosomal rearrangements

Duplication

Deletion

Inversion

Translocation

Operon

Parts of the Lac operon

Promoter = RNA Polymerase binding site

Operator = Repressor binding site

Constitutive expression

Gene is constantly expressed under normal cellular conditions

Positive vs Negative control

increase vs decrease gene expression

Upregulation vs downregulation

Cis vs trans-acting regulatory elements

Cis-acting = regulates adjacent gene on same chromosome

Trans-acting = regulates genes anywhere in genome via production of diffusible molecules

DNA-binding proteins aka Transcription factors

Inducible vs Repressible

Inducible = gene expression usually off

Repressible = gene expression usually on

How does the lac operon allow for environment-dependent digestion of lactose in bacteria?

LacI is constitutively expressed = repressor is constitutively produced

When no lactose present, inhibitor