Genetics Test 3

Sterile Insect Technique

used for the irradication of new world screw worm in the us

New world screw worm

• Recently a case was discovered in MD

• Irradication in the US in the 1960s using SIT

• Cases currently spreading north from south america

• Infects mostly cattle

• CRISPR/Cas 9 gene drive approach under development

Recombinant DNA Technology / genetic engineering

• Restriction enzymes

• Plasmid vectors

• PCR and cloning by PCR

DNA sequencing

• sanger

• next gen

Applications of molecular genetic techniques

• identification of variants in Genomes

• Transgenic organisms

• CRISPR genome editing

restriction enzymes / endonucleases

• recognize and cut DNA at specific nucleotie sequences

• Highly specific and highly efficient (cleave at speicific sequencences to make ds breaks)

• Different enzymes have different recognition sequences - recognition sequences are always palindromic

• Naturally occuring when used for defense against invading DNA by bacteria

Types of cuts generated by restriction enzymes

• staggered cuts (such as HINDIII)

  • Overhangs are complementary, which facilitates re-joining by DNA ligase

  • This allows us to paste in pieces of DNA by cutting them with the same restriction enzyme to generate recombinant DNA molecules

• blunt ends

Cloning genes

• getting lots of copies of an identical DNA sequence

3 basic features of cloning vector

• bacterial ori

• Drug resistance marker for selection

• Polylinker/MCS

Importance of bacterial ori in cloning vector

• must be recognized in host cell s that it is replicated along with the DNA that it carries

• this is so that we can get lots of copies of the DNA

Importance of drug resistance marker in cloning vector

• enable cells comtaining the vector to be identified

• way to kill off the cells that didn’t work

Importance of polylinker/mcs in cloning vector

• single cleavage site for each of the restriction enzymes used

• this gives versatility to paste in different DNA fragments

Steps to insertion of DNA fragment into vector

• digest plasmid with restriction enzyme

• remove 5’ phosphate ends using phosphatase so it doesn’t just close back up

• digest the foreign DNA with the same restriction enzyme

• Allow fragments to anneal due to sticky ends

• Join fragments back together using DNA ligase

• Transform bacteria

• Spread colonies on plate with ampicillin and isolate the colonies that grew

• Check if the cloning worked

Polymerase Chain Reaction (PCR)

• makes many copies of a DNA sequence of interest in vitro

• Super duper sensitive - can amplify a sequence from a tiny bit of DNA

• Requirements: heat stable DNA polymerase, thermocycler, DNA sample, primers (so you gotta know a little bit of the sequence already so you can put those on)

• Earned Mullis and Smith the nobel prize in 1993

Steps of PCR

• DNA is heated to 90-100 C to separate the strands

• DNA is cooled to 30-65 C to anneal primers

• DNA is heated to 60-70 C to let DNA polymerase synthesize

• Repeat

How transgenic crops are generated

• Foreign DNA (Bt gene) is inserted into a plasmid vector

• Transferred to Agrobacterium tumefaciencs

• Agrobacterium infects the crop

• The plasmid vector along with the DNA that it carriers is transferred into the plant cell, where it integrates into the plant chromosome

Genetically Modified Organism (GMO)

• genetic modification is done in lab setting as opposed to breeding

• Specific gene from a different species is added, generating a transgenic organism

Human genome

• 3 billion base pairs

• 30,000 genes, each gene generates about 3 proteins

• 0.1% difference between individuals

DNA fingerprinting

• used for identifying individuals

• uses known regions of the human genome that have expanded over time due to expansion of repeated sequences

• Variability in number of repeats at STR loci allows identification of individuals

• CODIS (combined DNA index system) uses 13 primers representing 13 unlinked loci

functional genomics

• characterizes what the sequences do

transcriptome

• all the RNA molecules transcribed from a genome

proteome

• all the proteins encoded by the genome

RNA sequencing

• getting seuqnece of all the mRNA in a certain cell, organisms, condition

• when, where, and under what condidtions different genes are expressed in different cells or organisms

• Can provide clues to function, espeicially when using a systems biology approach

haplotype

• specific set of genetic variants observed on a single chromosome or part of a chromosome

• each one is made up of a particular set of elles at each SNP

transposable element

• DNA sequences that can move about in the genome

• Mobile DNA, AKA transposon/jumping gene, can insert at many different locations in the genome

• Disocvered by barbara mcclintock

• Cause mutations by inserting into genes and promoting DNA rearrangements

transposase

• generates breaks in chromosomal DNA similar to restriction enzymes

Replicative transposition

• a new copy of the transposable element inserts in a new location, and the old copy stays behind

• generates more insertions over time

Nonreplicative transposition

• the old copy excises from the old site and moves to a new site

RNA intermediate transpotision

• requires reverse transcription to integrate into the target site

chromosome variation

• small scale variation visible as genome sequence

• Large scale differences like chromosome number and chromosome structure

CNVs arise from

• unequal crossing over

• olfactory receptors are an example

Polyploidy

• Changes in number of complete sets of chromosomes

• Common in plants

• No known examples of mammal viability

Aneuploidy

• changes in numbers of individual chromosomes

Allopolyploid

• chromosome sets from 2 (or more) species

• Generated by hybidization/crosses between speicies

Autopolyploid

• chromosome sets from the same species

• generated during mitosis (failure of cell separation → autotetraploid) or meiosis (nondisjunciton events → autotriploid)

• can generate a particular tissue that is polyploid

Autotriploids

• come from nondisjunction events in meiosis

• sterile

• causes seedless plants

Causes of aneuploidy

• deletion of centromere during mitosis and meiosis, leading to chromosome loss

• translocation followed by loss of small chromosome

• nondisjunction during mitosis or meiosis

Nullisomy

• type of aneuploidy where loss of both members of a homologous pair of chromosomes

• 2n-2

monosomy

• type of aneuploidy where loss of a single chromosome

• 2n-1

Trisomy

• type of aneuploidy where gain of a single chromosome

• 2n+1

tetrasomy

• type of aneuploidy where gain of two homologous chromosomes

• 2n+2

Familial down syndrome

• translocation of chromosome 21 onto another chromosome

Primary down syndrome

• random nondisjunction in egg formation causes trisomy 21

chromosome duplication

• segment of the chromosome is duplicated

chromosome deletion

• segment of the chromosome is deleted

chromosome inversion

• a segment of the chromosome is turned 180 degrees

translocation

• a segement of a chromosome moves from one chromosome to a nonhomologous chromosome or to another place on the same chromsome

• reciprocal or nonreciprocal

heterozygosity for deletion

• seen as a chromosomal loop

• can be viable if one copy is enough

• can produce phenotypic effects like an imbalance of gene products, bringing forth recessive mutations, or haploinsufficiency which is like a dosage effect

Pericentric inversion

includes the centromere

paracentric inversion

does not include the centromere

heterozygote for inversion

• loop forms when chromosomes align

• crossing over generates inviable gametes, and therefore inversions suppress recombination