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OLEMISS / Biology / BIOL 336 / What is the meaning of recombinant dna?

What is the meaning of recombinant dna?

What is the meaning of recombinant dna?

Description

School: University of Mississippi
Department: Biology
Course: Genetics
Professor: Ryan garrick
Term: Fall 2016
Tags: Genetics
Cost: 25
Name: Genetics Week 4 Notes
Description: Re-Upload... These notes cover chapters 17 and 18 lecture notes as well as notes on the scientific article we talked about in class.
Uploaded: 09/23/2016
11 Pages 47 Views 2 Unlocks
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Lecture 10


What is the meaning of recombinant dna?



9/16

Recombinant DNA

- Combining DNA that is from different organisms  

o Do not occur naturally

o Not a product of crossing over

 Ex: gene from fruit fly in bacteria  

• Gene from two different species that would not normally mate to create  mosaic-like chromosomes  

- Can also be used to isolate and copy specific DNA

Recombinant DNA

• Restriction enzymes: use recognition sites to cut DNA into fragments • Vectors/plasmids: takes up the cut DNA and incorporates it into its own by  sealing the sticky ends

• Competent bacterial cells: have the ability to take up the vectors/plasmids - Receptor sites – take plasmids up and bring them into cell so that DNA  replication can proceed inside of the competent cell  


What is the meaning of restriction enzymes?



Restriction Enzymes (REs)

- Original source from bacteria as a defense mechanism against  bacteriophages.  

• Scans and cuts DNA at both strands

- Cuts in zigzag fashion

• The fragments are now called restriction fragments 

- Single stranded DNA overhang creates “sticky ends”

Vectors (= plasmids)

• Transports the small DNA fragment into a competent cell  - Helps replicate inserted DNA  

Good vectors… (specific properties for easier cloning) - Polylinker region – region that would be cut by enzymes so that it  opens up to insert DNA fragment of target gene in it We also discuss several other topics like What is the goal of the humanistic approach?

o it is versatile and can be cut by many different enzymes o the region is a functional gene called Lac Z gene and is involved  in breaking down food and changes color of cell when it is  function ing properly


What is the meaning of the polylinker region?



 only works when NO inserted DNA in genes  

 bacterial cell with this plasmid will break down food and  turn blue  

- ampicillin resistance gene – bacteria takes this up and will be able to  grow on a medium  Don't forget about the age old question of What is the function of blood in human beings?

o allows bacteria cells to survive on a growth medium

• Replicate

• Are versatile because they have many cut sites for RE

• Transformed hosts can survive on the growth medium/go on to make more  copies If you want to learn more check out How does opportunity cost factor into comparative advantage?

Sticky ends and DNA ligase

FIG. 17-2

• Start with cleavage with EcoRI from eukaryotic gene and one from bacterial plasmid (vector)

• Fragments with complementary Tails

• Annealing allows recombinant DNA molecules to form by complementary  base pairing  

• DNA ligase seals the gap  

A recombinant DNA molecule

• If the DNA molecule took up the DNA successfully, blue colonies will form  - if unsuccessful or broken, white colonies will form  

• host cell needs the ampicillin resistant gene to survive ampicillin treated  growth medium and divide so that colonies will form  

What would happen if one of this vector failed to take up the plasmids at all?  - They would die before they’d even be able to be seen because they  would not be resistant to ampicillin and would die from the growth  medium  

Transforming the Host Cell

• E. coli is a good bacterial host/competent cell  

• competent cells will take up the DNA when electrically or heat shocked  - Heat shocked in lab?  

• yeast cells can be used as host cells to study eukaryotic genes

Polymerase Chain Reaction (PCR)

• Taq Polymerase is the DNA polymerase used If you want to learn more check out When does slavery demolish?

- Needs high temps  

- Very accurate  

• 3 steps – denature, anneal, extend

- Makes lots of copies of the DNA

• primers help set boundaries  

PCR: first cycle

• stress the dsDNA with high temperatures to Denature and split the strands  into 2 ssDNA

- 95°C

- each strand acts as template strand

• Primers anneal to each ssDNA strand at around 50°C

- Primers are fairly short and polar

o 3’ and 5’ ends

o what TAQ polymerase adds base pairs to  

• Taq adds base pairs to the 3’ of the primer (5’ of DNA complementary  strand)  

• Each cycle doubles the number of templates for the next cycle  - start with 2 strands and go through 8 cycles  

o 28 = 256 strands

 exponential increase

Advantages (v. Cloning)

• host cells are not needed

• takes way less time  

• More versatile due to primers because it creates more cut sites for DNA  polymerase If you want to learn more check out What are fats?

• Not a lot of DNA is necessary  

Limitations (v. cloning)  

• primers need some baseline info about the organism’s DNA  • Contamination is easy  

- Can accidentally clone DNA from a different source; need to be  cautious you are not amplifying your own DNA

Lecture 11

9/19

Cloning: recap

• cloning isn’t always perfect, sometimes the bacteria fails to take up the  inserted target DNA

- If something goes horribly wrong – all those bacterial cells will fail to  grow because they lack the gene that makes them resistant to  ampicillin  

Steps are involved in PCR and in correct order:  

- Denature –> annealing –> extension

More applications…  

• organism identification for illegal activities such as whale meat sales to  food industry  

• Chromosome walking  

- Essentially start at a particular place on a chromosome and extend  outward; generating an entire chromosome sequence  

• finding DNA mutations like when you use PCR

Following cloning or PCR

• Restriction mapping 

- Many different restriction enzymes to cut specific DNA into desired  fragments

- separate fragments using electrophoresis and other methods • Now can find out information from the cut sitesDon't forget about the age old question of What is the meaning of physiological stress response?

- how many cut sites

- order of cutting

- distance between the cutting

• restriction fragment length polymorphism (RFLPs) helps us identify DNA  variation

Electrophoresis

• Technique for separating DNA fragments according to differences in size  using a gel to act as a viscous matrix as well as an electric charge to pass  through this gel

- Send electric current from negative –> positive end; DNA migrates  towards positive end (Anode)

- Bands with longer fragments will take more time to move through the  gel

- Bands with shorter fragments will take less time  

Medical Diagnosis  

• RFLP analysis can help find disease-associated alleles in a genome  • Ex: Sickle cell

- A substitution mutation causes a different amino acid sequence which  leads to a ß-globin protein and  

- Homozygous: detrimental; heterozygous: effects show later  • A change in restriction enzyme recognition sites cause a new banding  pattern which can be used for diagnosing the disease the pattern represents  

Pharmacogenomics

• A lot of medications have common and/or fatal side-effects because  medication is variable to work for everyone…usually on works for about 60%  of the population

• Pharmacogenomics: selecting drugs and dosage based on the individual’s  genetic makeup  

- Individuals with same disease metabolize 6 MP differently  - More dependable than trial and error

- Differences due to genetic make-up (TPMT genotype)

- Genetic assay allows customized treatment

Complimentary DNA (cDNA)

• cDNA: only the DNA that codes for some specific protein  1. Extract mRNA

2. Convert mRNA back into its complimentary DNA using Reverse  transcription PCR

Reverse Transcription PCR

• In Reverse Transcription, mature mRNA is what we use to make templates  for coding  

• Oligo-DT primer will anneal to the 3’ end of the double stranded molecule

• Reverse transcriptase enzyme makes complementary DNA by extended  from the Oligo-DT primer

• a hybrid molecule of both mRNA and DNA is created so RNAse H enzyme  will digest that mRNA strand

• DNA polymerase will be used by any of the remaining mRNA  • the now double-strand of DNA acts as a snapshot of all the coding DNA in  the genome

DNA Sequencing

• How to determine how genes are organized by nucleotides  - Help determine genetic differences between organisms • Used to only be done by chain termination sequencing, or the Sanger  method.  

Chain Termination Sequencer  

• lots of colored peaks, one for each nucleotide, from a chromatogram  

• reads roughly 800 bp at a time

• a slow and expensive method compared to those newer methods

Lecture 12

9/21

“Next-generation” Sequencers

• Illumina Genome Analyzer 

- Faster and cheaper

o Reads around 150 bp

• Roche/454 GS FLX –

- Also faster and cheaper and reads around 400 bp per read.  ** relevant to align and assemble nucleotide sequences**

Ch. 18

Genomics and Bioinformatics

New-ish fields

** Review genomics, proteomics, and bioinformatics  

Genomics

• The study of the complete genetic information of small creatures that live  in the depths of the earth, who guard buried treasure

Organism

Diploid number (2n)

African Wild Dog 

78

Badger

32

Carp

104

Adders-Tongue

1262 (!)

Cotton 52

Karyotype is highly variable

Organismal “complexity”

• Organsimal complexity is not proportional to the number of chromosomes  or to genome size  

(absolute number of base pairs)

- Genome size – total amount of DNA (base pairs) within a haploid  genome  

Genome size also variable  

Organism

Billion bp (1000 million)

African Wild Dog 

2.66

Snapping Shrimp 

15.45

Carp

1.61

Poison Dart frog

8.70

Hugh-man

(human)

3.03

relate to absolute size of genome?

How does number of chromosomes  

- Size of African wild dog genome is quite larger than carps’ - Chromosomes are of different size (African Wild Dog’s are larger than  carps)  

- Frogs have a very large genome size compared to humans

Sequencing a Genome

• Genome – complete haploid set of all the DNA in a cell

- Not consistent size across organsism  

- Viruses have the smallest genomes, prokaryotes have median size,  and eukaryotes usually have the largest genomes  

• DNA sequencing  

- “shotgun” sequencing

o great for whole genome sequencing  

 Fragment the genome using restriction enzymes

 Sequence the fragments

 Assemble/order the pieces using some sort of  

electrophoresis  

Genomic Libraries

• Basically a whole genome that has been fragmented that can be inserted  and stored inside of vectors

• Does not involve PCR  

• Just get one copy of each sequence using the fewest number of fragments - BAC or YAC clones

Shotgun Sequencing Technique

• Fragment the genome

- Use specific REs one at a time  

• Get combinations of fragments  

• Computationally sequence the fragments

• End up assembling a whole chromosome sequence

Assembling “contigs”

• Contig – stretch of DNA sequences within one gene and across adjacent  genes that overlap

HP Computing (align, assemble)

• de novo alignment and assembly

- Not a lot of baseline data  

• Resequencing  

- Some baseline information  

Diploid Genomes

• heterozygous genotypes are possible so it may be necessary to align the  gene variants  

- Allele 1 v. Allele 2

DNA Sequence Alignment

• Scenario 1 – the reads do not start in same place

- Align sequences to see a match-up

- Mismatch between 2 nucleotides – point mutation/DNA sequence  substitution

- They are not identical  

Gene Pools

• Population – a gene pool can contain many allelic variants  • Might have to align the different variants of a gene  

- Look out for different kinds of polymorphisms

DNA Sequence Alignment

• Scenario 2 – many individuals and probably many alleles - Align sequences to see a match-up

o At least 5 different alleles  

 Take multiple sequence alignment and count the number of different sequences that exist

• Bioinformatics allows us to figure all of this quickly  

Annotating a Genome

• Need to identify genes with names and locations to interpret

Protein-coding genes have some hallmarks:

- Start codons for mRNA include AUG and for DNA = TAC o T pairs with A, A pairs with U, and C pairs with G <– in RNA - No stop codons until necessary so that a whole reading frame is  created

- Regulatory region upstream from primer  

o TATA-box

o Binding sites – often have conserved genetic DNA sequences

Can also compare to annotated sequences in databases:  

- BLAST searches can help identify sequences using unknown stretch of  DNA or amino acid sequences using the NCBI database to find the  closest match

o Query data base with stretch of DNA or amino acid sequence and find the “best” match that exists in data base… can infer the  likely chromosome or origin or function of the portion of DNA that is included in the search

Public databases

• National Center for Biotechnology Information (NCBI)

- Contains DNA sequences, annotated DNA sequences from whole  organisms and model organisms  

• Human chromosome maps, including known polymorphisms - Blue text – protein coding genes  

• Role of bioinformatics is not just to align and assemble existing short reads  of DNA sequences to generate whole genomes, but also in comparison of  whole genomes across individuals within same species or across different  species

BLAST Searches

• Query sequence –> stretch of sequence in which we generated and don’t  know its chromosomal origin or its function; going to compare it to its closest match in the blast search

- Best match highlighted in blue

- FIG 18-3

• Mouse sequence already annotated

- look for the matching portion

 an insulin receptor gene on chromosome 8

- Mouse = subject

• Rat (query) sequence was from an unknown genomic location • Now those identity and function can now be inferred based off of mouse  sequencing  

- Rat = query  

- Can infer that the 280 base pair sequence from the rat probably  originated from the same genomic location in the rat genome  - Can also infer that the 280 rat sequence does not contain stop codons  where they don’t belong if we expect this to be a protein coding  sequence…

o or if we were able to identify an AUG start codon or even an  upstream regulatory region  

Comparative Genomics

• covered so far

- how a whole genome sequence is produced

o shotgun sequencing – fragmenting, sequencing, and aligning and assembling

o annotate genome by identifying through inference what the  identity and function of DNA sequence is  

o once fully genome’d, compare it to the whole genome of a  second, third, or fourth species

• Compare different organisms and their genomes whether they are closely  related or not

- considerable similarity among organisms you would not have thought  • Helps scientists understand structure and function and expression of  human mutant diseases  

o comparative genomics provides a way to identify useful model  organisms that can help in experimental crosses that may inform us how to treat human genetic diseases

• Important evolutionary insights from comparative genomics - direct comparison between genome sizes between different classes of  organisms  

o prokaryotes, eukaryotes, viruses  

- Viruses – genome sizes small relatively; moderately strong correlation  between genome size and number of protein coding genes - Prokaryotes – strong linear relationship between genome size and  number of protein coding genes in the genome

- Eukaryotes – diffuse relationship (but still correlated) but not that tight; some large genomes with relatively few protein coding genes (large  portion of genome is comprised of non-coding DNA)

Lecture 13

9/23

From Article:  

• Adaptive change evolved rapidly (w/in 6,000 years)

• Predation rate increases for white mice in dark backgrounds and dark mice  in lighter backgrounds  

- Suggests that phenotype is adaptive and depends on the context of  the environment in which the mice lives

• Single DNA sequence change in protein coding portion – amino acid  replacement

- Different protein variant that functions differently

- How they figured out allelic variant with white coat colored is derived –  A SNP is 1 nucleotide along a stretch of protein coding region… this is  the only variant of focus that exists in natural populations…derived or  inherited?

o Used phylogenic approach  

o Derived because most mice in lineage is dark, only every now  and then a white phenotype will arise

o Tells us what the gene does

• Describes details to controlled cross and gives indication of what the  results mean

- Dominance/recessiveness

- 1 gene or more than 1

- gene is pleiotropic and RR is dark and CC is light so RC is intermediate  - copy of normal allele and copy of mutant allele

o look at figure 4

- proportion of chromosomes that carry the allelic variants - mosaic chromosomes – different phenotypes  

o 75 dominant phenotype and 25 recessive – standard Mendelian  assumption

 does not hold in this case (varying phenotypes)

 not always equal fitness

- Table 1 – shows results of the controlled cross focusing on the F2  generation and their phenotypes; genotypes have been sequenced o PVE is main column of interest because it shows the percentage  of variancts explained

• Figure 2 – basically what we get is 2 cell culture lines identical in every way except for which allelic variants of MC2R gene they have

- Alleles were expressed

- Figure shows how the 2 different cell lines perform with respect to  generating melanin

- MC1R has an activator which shows dark pigment and an antagonist  which produces a lighter color  

o Lots of activating protein – normal allele produces lots of melanin while mutant allele does not produce as much  

 Shows that DNA sequence mutation that alters amino acid  is encoded to the protein produced –> respond differently  in cell  

• The light coloration on the Gulf Coast is due to the MC1R allele while the  light coloration of the mice on Atlantic coast is not due to the MC1R allele.  This suggests that there are different ways to obtain that phenotype  depending on the environment of the population  

- White population on Atlantic coast evolved differently – nothing to do  with MC1R allele  

• Does evolutionary change proceed gradually through many small  mutational steps or can adaptation occur via a few large leaps? • Does adaptation generally proceed through dominant or recessive  mutations? Any of the above

• Do beneficial mutations tend ot affect protein function, or ists spatial or  temporal expression?  

• Are same genes and mutations responsible for similar traits in different  poulations or species

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