Molecular genetic analysis and biotech. DNA/RNA sequencing, quantitative analysis, stem cells and cloning

ch.19: pcr, cloning, dna sequencing

ch 21. (21.3) and ch.22 (22.1)

dna- deoxyribonucleic acid

PCR

amplify dna billion-fold in 2-3 hours

essentials:

  • two primers w sequences complementary to target region

  • DNA polymerase, usually Taq poly. (dna depedent dna poly.) enzyme

  • dNTPs (building blocks)

  • mg+ (acts as polymerase cofactor) -magnesium

molecular photocopying of dna. 30th cycle=2 bill copies

so dense you can use electrophoresis

Gel electrophoresis

used to determine if the target dna was in your samples, and confirm PCR product size

dna(- charge)—electric field turned on and on the other end w positive end.. little dna runs faster to (+) end. compare to known standard to observe

PCR: pros and cons

  • amplifies dna fragments w much less effort/time than cloning

  • requires prior knowledge of some part of the DNA region of interest in order to design primers (know genome)

  • Taq cannot proofread. Taq incorporates incorrect nucleotide every 20k bp

  • taq poly. can only amplify fragment sup to 2k bp

    • modifications can be made in pcr cycle ocnditions, but limit is 5k

recombinant DNA/ clones DNA

recombinant dna tech

  • also called genetic engineering

  • location, isolating, altering, and studying DNA segments

  • joining of genetic material from 2+ distinct sources

  • cloning segment of dna by using plasmid (molecular genetics)

cloning genes

  • origin or replication

    • ensures replication of vector

  • selectable markers

    • enables bacerial cells containing vector to be selected

  • one or more unique restriciton sites

    • allows that dna is cut into fewer pieces easier to piece together

restriction enzymes or restriction endonuclease are enzymes that cleaves dna into fragments at or near specific recognization sites within the molecule known as restriction sites

host dna is protected by a modification enzyme that modifies the prokaryotic dna and blocks cleavage

restriction modification system-one of the mech. that baceria and archaea use destroy invading dna like viruses. (enzymes)

restriction enzyme (RE) cutting is called re digestion and ligation of dan segments

ecoR1-cut

foreign dna insert or gene of interset

  1. cut out of donor dna use RE and pasted into plasmid dna

  2. pcr amipified form dna, w primers that have restriction sites on the ends, RE digested and ligated into plasmid

  3. pcr amplified form cDNA w primers that have restriction sites on the ends, RE digested and ligated into plasmid

ligations-dna joined togher using ligase as an enzyme

where does reverse transcription come from? Bacteria

rna dependent dna polymerase

scientistsi perform RT reactions to produce cDNA= complementary DNA

cDNA can be PCR amplified and cloned into DNA vectors (plasmids)

origin of repliation- selectable markers antibiotic resistance genes helps select baceria that were tranasformed

PCR products, clones dna, plasmids, gneomes are sequenced.

the beginning: sanger (dideoxy) sequencing-1977

  • chain termination method

  • dNT{ (extends dna strand) and ddNTP (terminates synthesis/ no H- no polymerization—chain termination)

now:

  • pcr w chianterminating ddntps w fluorochromes

  • run segments of diff. lengths through capillary electrophoresis

  • end up w chromatogram of sequence

  • used to sequence cloning sites, pcr products, etc.

  • chromatogram: higher peaks= more confident sequence results

next gen sequencing(NGS)

Stem Cells and Cloning

stem cells are undifferentiated bio cells capable of differentiating into forming every ell in a n organism; they are pluripotent.

anything development challenges them to be

two types of stem cells

  • embryoinic stem cells, which are isolated from the inner cell mass of blastocysts

  • adult stem cells, which are found in various tissues

    differentiated cells (intestinal cells)) divide by mitosis cellular phenotype is table and passed from one cell to another

Induced Pluripotent Stem Cells (iPSCs)

iPSCs- fully differentiated adult somatic cells, caused to dedifferentiate by treating with a “cocktail of 3 transcription facotrs” - effective for <1% cells

immortalized cell lines- pop. of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep doing division. they are not pluripotent

ex: HeLa cells- human cervical carcinoma cells. HeK293 cells from human embryoini cells

HeLa cells

  • 1st immortalized human cell line. form cervical cancer from Henrietta lacks in 1951

  • used to make 1st polio vaccine, salk vaccine

  • bills of vials of cells in labs

plant cells

totipotent cell: the cell that has the potential to develop into any cell types

cloning experiments on plants

cloning experiments on animals

many kinds of plants can be clones from isolated single cells

animal cloning

1997- dolly the sheep

1st somatic cell nuclear transfer (SCNT)

nucleous of somatic clel transfered to enucleated egg electrically stimulated to develop a transferred to surrogate

human- theraputic cloning