BIO; Genome projects & Genetic Technology

what is genome sequencing

involves determining the entire base sequence of an organism’s DNA

why’s genome sequencing important

allows to directly predict the proteome ( all proteins an organism can produce)

to identify potential antigens for creating vaccines against them

how is the simple organism’s(bacteria) proteome predicted from its genome

• possible bc they lack non coding DNA (e.g. introns)

• no regulatory genes affecting expression of other genes

so relationship between genome and proteome is straight forward

whys predicting the proteome from the genome of a a human more difficult

• large amounts of non coding DNA

• contain regulatory genes that affect the expression of other genes

what are the key advances in sequencing methods

increasingly rapid

cost effective

automated

whats recombinant DNA technology

involves transferring fragments of DNA from one organism to another

whats an transgenic organism

organism that receives the transferred fragment of DNA

why does recombinant DNA technology work

• bc the genetic code is universal;

meaning the same codons code for the same amino acids in all organisms

• the mechanisms for transcription&translation is also universal;

meaning inserted gene can be transcribed into mRNA and translated into a protein in the host cell

whats the first step of recombinant DNA technology

isolate the fragments of DNA (3 step process;)

1. reverse transcription

2. restriction endonuclease

3. gene machine

process of reverse transcription to produce DNA fragments

1. reverse transcriptase enzyme makes DNA copies from mRNA

2. cell that naturally produces protein of interest, is selected

3. these cells have large amounts of mRNA for this protein

4. reverse transcriptase enzyme joins DNA nucleotides with complimentary bases to mRNA sequence via condensation reactions of phosphodiester bonds

5. single stranded DNA is made (cDNA)

6. DNA polymerase is used to make this cDNA fragment double stranded

process of restriction endonuclease to produce DNA fragments

1. many restriction endonuclease (enzymes) have different recognition sequences (active site complimentary in shape to different DNA base sequences)

2. so each enzyme cuts the DNA at a specific location;

3. some enzymes cut at the same location in double strand - creates a blunt end

4. some enzymes cut at different locations - creates staggered ends (aka sticky ends)

why are sticky ends important

ability to join to DNA with complimentary base pairs

of the palindromic sequences

whats meant by palindromic

when the order of a sequence is the same forwards and backwards

process of gene machine in creating DNA fragments

1. identify amino acid sequence to work out mRNA and DNA sequence

2. DNA base sequence is entered into computer

3. computer checks for biosafety that DNA created is safe and ethical to produce

4. computer creates small sections of overlapping single strands of nucleotides that make a gene (oligonucleotides)

5. oligonucleotides join to create DNA for entire gene

advantage of using gene machines to create DNA fragments

makes intron free DNA;

bc prokaryotic cells dont have introns so can’t remove them via splicing

if intron free gene is put into bacteria cell then splicing is not required

two ways to clone DNA fragments

in vivo cloning

in vitro cloning

whats the promoter region of DNA

• a DNA base sequence placed before the gene

• acts as the binding site for RNA polymerase & transcription factors

• to initiate transcription of the gene

what the terminator region

• DNA base sequence placed after the gene

• signals the end of transcription

• ensures RNA polymerase to stop transcribing at the correct point

importance of promotor and terminator regions

without promotor - gene will not be transcribed

without terminator - transcription might continue into unwanted regions

whats a vector

something that carries the isolated DNA fragment into a host cell

e.g. plasmids

how to make recombinant DNA

1. plasmid(vector) is cut open using same restriction endonuclease (as one used to cut the DNA fragment)

2. this creates the same sticky ends in plasmid as the DNA fragment

3. enzyme ligase anneals (sticks) DNA fragment and plasmid together

ligase catalyses condensation reaction forming phosphodiester bonds between nucleotide

how to insert recombinant DNA into host cell (transformation of host cells)

1. host cells are mixed with Ca2+ and heat shocked (sudden increase in temp)

2. this increases the permeability of host cell’s cell membrane for plasmids w/ recombinant DNA to enter

what are the 3 issues that can occur that make it unable to identify transformed cells

• recombinant plasmid doesnt get inside the host cell

• plasmid rejoins before the DNA fragment enters

• DNA fragment sticks to itself, rather than inserting into the plasmid

how are transformed cells identified

using marker genes

what are marker genes

genes on the plasmid that can be used to identify which bacteria successfully took in the recombinant plasmid

what are the 3 different marker genes used

• antibiotic resistance genes (common application)

• genes coding for fluorescent proteins

• genes coding for enzymes

whats in vitro cloning

fragments of DNA can be amplified(copied) in vitro (outside cell) in lab/glass (e.g. PCR)

whats the PCR method

1. temp is increased to 95C - breaks hydrogen bonds and split DNA into single strands

2. temp is then decreased to 55C - so primers can attach (annealing)

3. DNA polymerase attaches complimentary free nucleotides

4. makes a new strand to align next to each template (synthesis)

5. temp increased to 72C for DNA polymerase

how to calculate PCR method to calculate how many DNA fragments are made

$$y\times2^{n}$$

n = number of rounds of division

y = number of DNA molecules you started with

advantages of PCR

• automated - more efficient

• rapid - 100 billion copies of DNA made within hours

• doesn't require living cells - quicker/less complex techniques needed

whats a DNA probe

• short single stranded piece of DNA

• that is complimentary to a specific allele or gene

probs are labelled with a radioactive isotope or a fluorescent marker to allow detection

how can a DNA probe be detected

detected using X ray film or UV light

presence of a probe indicates the presence of the target allele

whats DNA hybridisation

occurs when probe binds to its complimentary base sequence within a DNA sample

how is sample prepared for DNA hybridisation

1. DNA is extracted and heated to separate its double strand

2. probe is then added

3. if target sequence is present, the probe will bind to it via complimentary base pairing

what’s VNTRs

short sequences of bases repeated a different number of times (this differs from person to person)

these are highly useful in DNA analysis;

bc VNTR patterns are inherited and highly specific for identification

whats genetic finger printing

technique used to analyse DNA fragments containing VNTRs

whats the process of genetic finger printing

1. DNA is amplified using PCR to produce enough DNA for analysis

2. DNA is cut into fragments using restriction enzymes close to VNTR sequence

3. these fragments are separated by gel electrophoresis (sorts them by size)

4. alkaline added to separate double strands of DNA

5. DNA probe is added, which binds to specific VNTR sequences

6. this makes patterns of bands

7. which form genetic fingerprint which is unique to an individual (not in identical twins)

how does gel electrophoresis work in genetic fingerprinting

• DNA fragments are loaded into small wells of agar gel;

• this gel is placed in a buffer liquid with an electric voltage applied

• DNA is negatively charged so DNA samples move through the gel towards the positive end of the gel

DNA moves a certain distance through agar gel based on how many bases it contains

e.g. bigger DNA base sequence = heavier = short distance

e.g. shorter DNA = lighter = longer distance

5 key uses of genetic fingerprinting

1. paternity and family testing - compare child’s genetic fingerprint with parents (matching bands confirm biological relationships)

2. forensic science - matches in VNTR patterns can link a person to the scene

3. medical diagnosis - identify genetic markers for inherited diseases (helps early diagnosis for carriers and treatments)

4. animal &plant breeding - confirms parentage, ensures desirable traits are passed on

5. assessing genetic diversity - compares VNTR patterns in a population (greater diversity = adaptable population)