Kamila Balušíková. DNA – sequence of genes, repetitive sequence of noncoding regions RNA ...

Methods of DNA and RNA study

Kamila Balušíková

Diagnostics

DNA – sequence of genes, repetitive sequence of noncoding regions

RNA Proteins

gene expression

Source of nucleic acids

DNA of certain gene – all nuclear cells

RNA of certain gene – only the cells, where this gene is

expressed

DNA diagnostic

What can we detect ?

Monogenic and polygenic inherited diseases Some types of tumors Presence of infection (detection of pathogens) Disease progress during the therapy Identification of people in forensic medicine HLA-typization in cases of transplantation

… Prevention - examination: - preimplantation

- prenatal - presymptomatic

DNA polymorphisms

Variability in DNA sequence between different individuals of the same species

Allele polymorphisms physiological function, with frequency > 1%predisposition to polygenic diseases

Mutations pathological function, with frequency < 1%cause of monogenic diseases

Characteristics of DNA diagnostics

Detection of certain polymorphism of the predisposition gene

TARGET ANALYSES COMPLETE ANALYSES

Target analyses

The localization and the whole sequence of a gene is known

The mutation of the gene is known

An examination of family membersis not needed

Complete analyses

The localization and the whole sequence of a gene is known

Mutations of the gene are unknown

An examination of family members is necessary

Principle of testing

DNA isolation

PCR (amplification of a DNA region)+ other analyses

result visualization

DNA izolation

Basic steps:

› Cell lysis → DNA release› Protein removal

ProteaseAdsorption or extraction

› DNA precipitation by ethanol → impurities

removal› DNA dissolution in water or buffer

DNA purity and concentration

Spectrofotometryabsorption maximum

for nucleic acids 260 nmfor proteins 280 nm

→ DNA concentration: at 260 nm→ DNA purity is calculated by ratio 260/280 nm

DNA purity and concentration

Gel electrophoresis with fluorescent dyes (approximate)

› DNA is stained by intercalating dyes in gel › Gel is loaded with DNA standard

(its concentration is pre-evaluated) – comparison of two light intensities

Gel electrophoresis- separation method

Separating of DNA fragments (RNA, protein molecules) according to their molecular weight (size) Principle: movement of charged

molecules in electric field

Gel electrophoresis

the nucleic acids consist of negatively charged phosphate groups → → the movement direction goes from cathode (-) to anode (+)

The movement rate of DNA in gel depends on DNA fragment size in indirect proportion(the larger the slower)

Gel – sieve structure of polymer molecules with pores

agarose x polyacrylamide› Different resolving power:

polyacrylamide separates DNA fragments varying in single nucleotide in their lengths

agarose separates fragments which lengths differ minimally in 10 nucleotides (wider range – hundreds base pairs)

Nucleic acid visualization in gel

fluorescent dye is added to the gel (e.g. ethidium bromide)

› Intercalates into the DNA structure› After light exposure, its complex excites

photons (shines)

MW marker(bp = base pairs)

DNA fragments

PCR (polymerase chain reaction)

PRINCIPLE: multiplying (amplification) of selected DNA part(s)

Reaction is performed in cycles (30 – 40 cycles)

Each cycle consist of 3 steps (change of temperature is constant affects individual steps)

• denaturation• annealing• extension

PCR (polymerase chain reaction)

Basic compounds in PCR reaction

DNA sample Pair of primers Free nucleotides (dATP, dTTP, dCTP, dGTP) DNA polymerase with buffer

Primers

Short oligonucleotides (20 – 30 nucleotides)

Forward primer a reverse primer – one primer for one DNA strand

Complementarity to the sequences at the 3´end of corresponding DNA strand

Delimit the target DNA region which will be amplified

Their binding is influenced by temperature – depends on primers length and type of

nucleotides

Sugar-phosphate skeleton

base pairs bounded by hydrogen bounds

Steps of PCR

1.Denaturationbreaking of H-bounds in DNA double strand; separated strands are created (T > 94°C)

2.Annealingprimers connection to separated DNA strands (Tanneal. = ?)

3.Extension (elongation)new DNA strand synthesis; DNA polymerase synthesize new DNA strand according to the old (template) one (T = 72°C)

Temperature is a constant in each step

Amplification

Exponential function› Copies number of multiplying DNA region = 2n,

when n is number of cycles

first cycle(creating of two double stranded DNA

molecules)

second cycle(creating of four double stranded DNA

molecules)

third cycle(creating of eight double stranded DNA

molecules)

DNA synthesis

DNA synthesis

DNA synthesis

Separation of DNA strands and primer pairing

Separation of DNA strands and primer pairing

Separation of DNA strands and primer pairing

Target region of double stranded chromosomal DNA we want to amplify

PCR types

PCR with allele specific primers – target analyses(ASO-PCR = PCR with allele specific oligonucleotides)

PCR with general primers – followed by PCR product analysis – target, complete analyses

PCR subtypes

Nested PCR (includes two successive PCR reaction)

Multiplex PCR (employs two or more PCR in same time – one reaction mix)

PCR product analysis(PCR with general primers)

Unknown mutation – complete analyses

Sequencingsearching for complete (exact) order of nucleotides in amplified DNA fragment

PCR product analysis(PCR with general primers)

Known mutation – target analyses

Hybridization analysis of PCR product using labeled probe

RFLP (restriction fragment-length polymorphism)PCR product is specifically digested using restriction enzymes (restriction endonuclease – restrictase)

Gene expression levels – mRNA– proteins

mRNA – Real-Time PCR, Northern blot Proteins – Western blot

Detection of certain gene expression

Gene expression on mRNA level

Real-Time PCR → PCR for qualitative and quantitative analysis

(x DNA diagnostic – qualitative analysis only)

› We measure increasing amount of PCR product in time(how much?) in each cycle of PCR reaction

› RNA cDNA (complementary DNA)

Reverse transcription

Reverse transcriptase

Real-Time PCR

› When target gene is not expressed, mRNA is not created – no amplification

› The more of target gene mRNA, the more of cDNA, the faster is cDNA amplified → tested gen is more expressed than other gene or the same gene but under different conditions (comparative analysis)