Topic 7 flascards

Genome

All organisms DNA

PCR

Amplies DNA, cooling for primer binding and DNA synthesis repeated around 30 times

PCR

1. Reaction mixture is set up by mixing the DNA sample, primers and free nucleotides and tap polymerase

2. Heated to 95C to break H bonds

3. Cooled between 50-65 C annealing of primers

4. Increase 70c temp Taq p works at

5. Cycle repeats

What is taq polymerase

Heat stable DNA polymerase

DNA sequencing

uses fluorescent terminator nucleotides to create readable fragment chains, separated by gel electrophoresis.

DNA profiling

Used for forensics and paternity

Terminator nucleotide

Incorporated into a growing chain rep is terminated

DNA profiling method

1. Fragments cut with restriction endonucleases

2. Separated and visualised using gel e

3. Transferred via southern b

4. Gene probes added and bind w DNA (hybridisation)

5. Probed bind to repeated seqs (mini/micro satellites) to visualise patterns

Southern blot

Alkaline buffer solution, nylon filter - dry absorbent mat draws solution contains DNA fragments to filter - fragments visible as ‘blots’

Gene probes

Labelled complementary seqs that fluorescence or are radioactive

More closely related

= more sim repeats VNTRs are

Mini satellites/ VNTRs

Sequences of DNA in introns

Transcription factors

Bind to promotor/enhancer regions to activate/suppress transcription

Promoter regions

Enable RNA polymerase to bind = promote transcription

Enhancer regions

Change chromatin stucture reg DNA activity - more or less likely to open RNA p

• Open = activate gene exp

• Close = opp

Epigentics

• Heritable + reversible modifications change gene expression but not DNA seq

• DNA methylation

• Histone modification

DNA methylation

Add methyl group to CpG site of DNA

DNA methylation effect

• prevents transcription + makes histone stcryire more/less DNA accessible to RNA p

Histone modification

Acteylation = activation

Methylation = activation or repression

Acteylation

• Adds acetyl group COCH3

• Activates chromatin

• = transcription

Methylation

• Adds methyl group

• = inactivation or activation of chromatin depending on position of lysine

Non-coding RNA

Affects transcription

E.g. ncRNA coats 1 x chrom suoerocils + condenses = stable inactive Barr body to maintain balance of gene products

RNA splicing

• creates multiple proteins from one gene by removing introns and some exons.

Splicesosomes

• Join the exons together enzyme = way they are joined = mat RNA

Stem cells

Undifferentiated cells ability to divide into other types of cells

Multi potent

Limited

Pluripotent

• Give rise to may types of cells not embryonic

Totipotent

Give rise to all specialise cells including embryonic

time of totipotent

Limited

What stem cell is used for travel g human disorders

Pluripotent replace damaged tissue

Ethical issues of stem cells

Pros

• save lives

• Improve quality

Cons

• embryos killed in process unethical

• Risk of infection when cells are transplanted

Uses of stem cells

• diabetes

• MS

• Parkinson’s

Induced pluripotent cells IPSCs

reprogrammed adult cells, bypass ethical issues but may cause tumors.

How do iPSCs do this?

1. Fibroblasts taken from skin

2. Viruses used as vectors to introduce 4 gene ex for transcription

3. Factors = active g in adult cells = pluripotent cells

4. Cells sim to embryonic - effectiveness is unknown

I1st stage of recombinant DNA solation of Gene

• use restriction endonuclease = sticky ends exposed bases

• Reverse t = DNA from mRNA

2nd stage of recombinant DNA

• Cut plasmid w same restcition endoneucleade leave comp sticky ends

3rd stage

• Join plasmid with DNA ligase = recombinant DNA

4th

Reincorporate plasmid into host nucleus

Vectors types

• Plasmids

• Viruses

• Liposomes

• Gene guns

• Micro injection

Effective vectors

• Target right cells

• Incorporate gene into host genome

• Have no adverse side effects

Gene markers

Fluorescence or anti idiotic r help identify modified cells

What are knockout mice?

Mice with specific genes disabled to study function.

What are transgenic plants?

Plants with genes from unrelated organisms.

Gen m using agrobacterium tumefaciens

• Ti plasmid extracted

• Bact genes inserted into plasmid via gen m

• Plasmid returns to bacterium

• Plant infected with bact

• Grows a crown gall

Uses of gene mod

• Flood r

• Pesticide production

• Herbicide r

• Changing nutrients value

Soya beans exp

• Linoleic acid ( poly)

• Replaced by oldie mono

  • more healthier

What are 4 genes involved in inducing pluripotent

Oct3/4, Sox2, c-Myc, and Klf4